Method of distinguishing an IBRV-vaccinated bovine from a bovine infected with a wild type virus

ABSTRACT

The present invention provides recombinant infectious bovine rhinotracheitis (IBR) viruses useful in vaccines to protect bovines from infectious bovine rhinotracheitis and other bovine diseases. The present invention further provides methods for distinguishing an animal vaccinated with the vaccine of the present invention from an animal infected with a naturally-occurring IBR virus. The present invention also provides isolated DNA encoding the gpE glycoprotein of IBR virus and isolated DNA encoding the gpG glycoprotein of IBR virus.

This application is a continuation of U.S. Ser. No. 08/247,475, filed May 23, 1994, now U.S. Pat. No. 5,593,873, which is a continuation of U.S. Ser. No. 07/732,584, filed Jul. 18, 1991, now abandoned, which is a continuation-in-part of U.S. Ser. No. 07/696,262, filed Apr. 30, 1991, now abandoned, which is a continuation of U.S. Ser. No. 06/933,107, filed Nov. 20, 1986, now abandoned, and a continuation-in-part of U.S. Ser. No. 07/649,380, filed Jan. 31, 1991, now abandoned, which is a continuation of U.S. Ser. No. 07/078,519, filed Jul. 27, 1987, now abandoned, and a continuation-in-part of U.S. Ser. No. 07/225,032, filed Jul. 27, 1988, now U.S. Pat. No. 5,223,424, and a continuation-in-part of U.S. Ser. No. 06/823,102, filed Jan. 27, 1986, now U.S. Pat. No. 5,068,192, and a continuation-in-part of U.S. Ser. No. 07/192,866, filed May 11, 1988, now U.S. Pat. No. 5,047,237, the contents of which are hereby incorporated by reference.

Within this application several publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention involves recombinant infectious bovine rhinotracheitis (IBR) viruses useful in vaccines to protect bovines from naturally-occurring infectious bovine rhinotracheitis virus and other bovine diseases.

BACKGROUND OF THE INVENTION

The ability to isolate viral DNA and clone this isolated DNA into bacterial plasmids has greatly expanded the approaches available to make viral vaccines. The methods used to make the present invention involve modifying cloned viral DNA sequences by insertions, deletions and single or multiple base changes. The modified DNA is then reinserted into the viral genome to render the virus non-pathogenic. The resulting live virus may then be used in a vaccine to elicit an immune response in a host animal and to protect the animal against a disease.

One group of animal viruses, the herpesviruses or Herpetoviridae, is an example of a class of viruses amenable to this approach. These viruses contain 100,000 to 200,000 base pairs of DNA as their genetic material. Importantly, several regions of the genome have been identified that are nonessential for the replication of virus in vitro in cell culture. Modifications in these regions of the DNA may lower the pathogenicity of the virus, i.e., attenuate the virus. For example, inactivation of the thymidine kinase gene renders human herpes simplex virus non-pathogenic (28), and pseudorabies virus of swine non-pathogenic (29).

Removal of part of the repeat region renders human herpes simplex virus non-pathogenic (30,31). A repeat region has been identified in Marek's disease virus that is associated with viral oncogenicity (32). A region in herpesvirus saimiri has similarly been correlated with oncogenicity (33). Removal of part of the repeat region renders pseudorabies virus non-pathogenic (U.S. Pat. No. 4,877,737, issued Oct. 31, 1989). A region in pseudorabies virus has been shown to be deleted in naturally-occurring vaccine strains (11,3) and it has been shown that these deletions are at least partly responsible for the lack of pathogenicity of these strains.

It is generally agreed that herpesviruses contain non-essential regions of DNA in various parts of the genome, and that modifications of these regions can attenuate the virus, leading to a non-pathogenic strain from which a vaccine may be derived. The degree of attenuation of the virus is important to the utility of the virus as a vaccine. Deletions which cause too much attenuation of the virus will result in a vaccine that fails to elicit an adequate immune response. Although several examples of attenuating deletions are known, the appropriate combination of deletions is not readily apparent.

Infectious bovine rhinotracheitis (IBR) virus, an alphaherpesvirus with a class D genome, is an important pathogen of cattle. It has been associated with respiratory, ocular, reproductive, central nervous system, enteric, neonatal, and dermal diseases (34). Cattle are the normal hosts of IBR virus, however it also infects goats, swine, water buffalo, wildebeest, mink, and ferrets. Experimental infections have been established in muledeer, goats, swine, ferrets, and rabbits (35).

Conventional modified live virus vaccines have been widely used to control diseases caused by IBR virus. However, these vaccine viruses may revert to virulence. More recently, killed virus IBR vaccines have been used, but their efficacy appears to be marginal.

IBR virus has been analyzed at the molecular level as reviewed in Ludwig (36). A restriction map of the genome is available in this reference, which will aid in the genetic engineering of IBR according to the methods provided by the present invention.

As reported in the current literature, IBR virus has been engineered to contain a thymidine kinase deletion (43,44) and a deletion in the gIII gene (45,46). However, no evidence has been presented for the deletions in the US2, repeat, gpG, or gpE regions. In the subject application, we demonstrate the usefulness of such deletions for both the attenuation of IBR virus and for the development of gene deleted marker vaccines.

As with other herpesviruses, IBR virus can become latent in healthy animals which makes them potential carriers of the virus. For this reason it is clearly advantageous to be able to distinguish animals vaccinated with non-virulent virus from animals infected with disease-causing wild type virus. The development of differential vaccines and companion diagnostic tests has proven valuable in the management of pseudorabies disease (47). A similar differential marker vaccine would be of great value in the management of IBR disease. The construction of differential diagnostics has focused on the deletion of glycoproteins. Theoretically, the glycoprotein chosen to be the diagnostic marker should have the following characteristics: (1) the glycoprotein and its gene should be non-essential for the production of infectious virus in tissue culture; (2) the glycoprotein should elicit a major serological response in the animal; and (3) the glycoprotein should not be one that makes a significant contribution to the protective immunity. Four major IBR virus glycoproteins (gI, gII, gIII, and gIV) have been described in the literature (48). Three of these genes, gI, gIII, and gIV, have been sequenced and shown to be homologous to the HSV glycoproteins gB, gC, and gD, respectively. Although it has been suggested that the gII protein is analogous to HSV gE, no sequence evidence has been presented to confirm that suggestion (48). The gB and gD homologues are essential genes and would not be appropriate as deletion marker genes. The gC gene of herpesviruses has been shown to make a significant contribution to protective immunity as a target of neutralizing antibody (49) and as a target of cell-mediated immunity (50). Therefore, the gC gene is not desirable as a deletion marker gene. As indicated above, Kit et al. (45) have described the deletion of the IBR virus gIII as a marker gene. It would be expected that such a deletion would compromise the efficacy of an IBR vaccine.

For pseudorabies virus (PRV) the criteria for a deletion marker gene are best met by the glycoprotein X (51). Wirth et al. (52) suggests the existence of a "gX homologue of HSV-1" in the IBR virus. It is not clear what is meant by this because although there is a PRV gX gene, there is no reported HSV-1 gX gene or gX homologous gene. In any case, no sequence evidence is presented to support this suggestion. We present clear evidence of homologues of PRV gX (HSV-2 gG) and PRV gI (HSV gE) in IBR virus and demonstrate their usefulness as diagnostic markers.

The present invention provides a method of producing a fetal-safe, live recombinant IBR virus which comprises treating viral DNA from naturally-occurring live IBR virus so as to delete from the virus DNA corresponding to the US2 region of the naturally-occurring IBR virus. The present invention also provides viruses in which (1) DNA corresponding to the US2 region of naturally-occurring IBR virus has been deleted, and (2) DNA encoding gpG and/or gpE has been altered or deleted. Such viruses are useful in vaccines which need diagnostic markers and are safe for use in pregnant animals.

The ability to engineer DNA viruses with large genomes, such as vaccinia virus and the herpesvirues, has led to the finding that these recombinant viruses can be used as vectors to deliver immunogens to animals (53). The herpesviruses are attractive candidates for development as vectors because their host range is primarily limited to a single target species (54), and they have the capacity for establishing a latent infection (55) that could provide for stable in vivo expression of a desired cloned polypeptide. Herpesviruses have been engineered to express a variety of foreign gene products, such as bovine growth hormone (56), human tissue plasminogen activator (57), and E. coli β-galactosidase (58,59). In addition, possible immunogenic polypeptides have been expressed by herpesviruses. Whealy et al. (60) expressed portions of the human immunodeficiency virus type 1 envelope glycoprotein in pseudorabies virus (PRV) as fusions to the PRV glycoprotein III. The hepatitis B virus surface antigen (61) and a hybrid human malaria antigen from Plasmodium falciparum have been expressed in herpes simplex virus type 1 (HSV-1) (62). The IBR viruses described above may be used as vectors for the insertion of genes encoding antigens from microorganisms causing important cattle diseases. Such recombinant viruses would be multivalent vaccines protecting against IBR as well as other diseases. Kit et al. (63) have described the expression of a Foot and Mouth disease antigen in IBR virus. In some of the prior applications from which the subject application claims priority (which precedes the Kit publication by at least three years), we described the use of IBR virus to express several foreign genes including the E.coli β-galactosidase (lacZ) gene, the TN5 neomycin resistance gene, and antigens from bovine rota virus, and parainfluenza-3 virus (see U.S. Ser. No. 06/933,107, filed Nov. 20, 1986, now abandoned, and U.S. Ser. No. 07/078,519, filed Jul. 27, 1987, now abandoned). These applications precede the Kit publication by at least three years. The viruses described in this application provide a combination of attenuation, differentiation and multivalency. These properties make such viruses useful as vaccines for the management of cattle diseases.

SUMMARY OF THE INVENTION

The present invention provides recombinant infectious bovine rhinotracheitis (IBR) viruses useful in vaccines to protect bovines from infectious bovine rhinotracheitis and other bovine diseases. The present invention further provides methods for distinguishing an animal vaccinated with the vaccine of the present invention from an animal infected with a naturally-occurring IBR virus. The present invention also provides isolated DNA encoding the gpE glycoprotein of IBR virus and isolated DNA encoding the gpG glycoprotein of IBR virus. The present invention also provides a method of producing a fetal-safe, live recombinant IBR virus which comprises treating viral DNA from a naturally-occurring live IBR virus so as to delete from the virus DNA corresponding to the US2 region of the naturally-occurring IBR virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Details of the IBR Cooper Strain. Diagram of IBR genomic DNA showing the unique long, internal repeat, unique short, and Terminal repeat regions. Restriction maps for the enzymes HindIII, EcoRI, and XbaI are indicated (7). Fragments are lettered in order of decreasing size. The unique short region is also expanded for inclusion of more detail. The location of several genes is also indicated, they are unique short 2 (US2), immediate early genes (IE) (20), glycoprotein G (gpG), glycoprotein IV (gpIV) (17), glycoprotein E (gpE). Note that due to the inversion of the short region, which includes the unique short, internal, and terminal repeats, four half molar HindIII fragments are present (HindIII D, C, F, and H).

FIG. 2 Details of S-IBR-002. Diagram of S-IBR-002 genomic DNA showing the unique long, internal repeat, unique short, and Terminal repeat regions. Restriction maps for the enzymes HindIII, EcoRI, and XbaI are indicated (7). Fragments are lettered in order of decreasing size. The EcoRI B and F fragments are expanded for inclusion of more detail. The ⁻ 800 BP repeat deletions are indicated by deltas. Note that due to the inversion of the short region, which includes the unique short, internal, and terminal repeats, four half molar HindIII fragments are present (HindIII D, C, F, and H).

FIG. 3 SEQ ID NO: 1 DNA sequence of the IBR Unique Short 2 gene. The sequence of the first 1080 base pairs of the HindIII K fragment, reading from the HindIII K/HindIII O junction, are shown. The unique short 2 (US2) gene is transcribed toward the HindIII K/HindIII O junction as indicated in FIG. 1. The sequence has been reversed and complemented in order to show the translation start and termination of the US2 gene.

FIGS. 4A and 4B SEQ ID NO: 2-6 Homology between the IBR US2 (SEQ ID NO: 2) protein and the US2 proteins of HSV-1 (SEQ ID NO: 3), PRV(SEQ ID NO: 4), HSV-2 (SEQ ID NO: 5), and MDV(SEQ ID NO: 6). (FIG. 4A) Matrix plot of the amino acid sequence of the IBR US2 protein (309) against the amino acid sequence of the HSV-1 US2 protein (291) (8). (FIG. 4B) Alignment of a conserved region between IBR US2 protein, HSV-1 US2 protein, PRV US2 protein (256 amino acids) (21), HSV-2 US2 protein (291) (9), and MDV US2 protein (270 amino acids) (1).

FIG. 5 SEQ ID NOS: 7-9 Details of the Nasalgen deletion. Diagram of IBR genomic DNA showing the unique long, internal repeat, unique short, and terminal repeat regions. A restriction map for the enzyme HindIII is indicated. Fragments are lettered in order of decreasing size. The unique short region is expanded for inclusion of more detail. The location of the deletion in the Nasalgen HindIII K fragment is shown. Three regions of DNA sequence are also shown. The first line (SEQ ID NO: 7)shows the first 60 base pairs upstream of the HindIII O/HindIII D junction in the IBR Cooper strain. The second line (SEQ ID NO: 8) shows the first 60 base pairs upstream of the HindIII K/HindIII D junction in the Nasalgen strain. The third line (SEQ ID NO: 9) shows 60 base pairs flanking the DNA encoding amino acid 59 of the IBR US2 gene in the IBR Cooper strain.

FIG. 6 Details of S-IBR-027. Diagram of S-IBR-027 genomic DNA showing the unique long, internal repeat, unique short, and terminal repeat regions. Restriction maps for the enzymes HindIII, EcoRI, and XbaI are indicated (7). Fragments are lettered in order of decreasing size. The unique short region is also expanded for inclusion of more detail. The location of several genes is also indicated, they are unique short 2 (US2), immediate early genes (IE) (20), glycoprotein G (gpG), glycoprotein IV (gpIV) (17), glycoprotein E (gpE). The unique short region and repeat region deletions are indicated by deltas. The location of the approximately 1200 BP deletion of the US2 gene is shown in the expanded region. Note that due to the inversion of the short region, which includes the unique short, internal, and terminal repeats, four half molar HindIII fragments are present (HindIII D, C, F, and H).

FIG. 7 SEQ ID NOS: 10-15 Detailed description of the DNA insertion in Homology Vector 129-71.5. Diagram showing the orientation of DNA fragments assembled in plasmid 129-71.5. The origin of each fragment is indicated in the table. The sequences (SEQ ID NOS: 10-15) located at each of the junctions between fragments is also shown. The restriction sites used to generate each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, !, indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: polyadenylation signal (pA), infectious bovine rhinotracheitis virus (IBR), Herpes simplex virus type 1 (HSV-1), thymidine kinase (TK), neomycin resistance (NEO), bacterial transposon Tn5 (Tn5).

FIG. 8 SEQ ID NO: 16 DNA sequence of the IBR glycoprotein G gene. The sequence of approximately 1400 base pairs of the HindIII K fragment, starting approximately 2800 base pairs downstream of the HindIII K/HindIII O junction, are shown. The glycoprotein G (gpG) gene is transcribed away from the HindIII K/HindIII O junction as indicated in FIG. 1. The translational start and termination of the gpG gene are indicated.

FIGS. 9A and 9B SEQ ID NOS: 17-19 Homology between the IBR gpG (SEQ ID NO: 17) protein, the gpX protein of PRV (SEQ ID NO: 18) and the gpG protein of HSV-2 (SEQ ID NO: 19). (FIG. 9A) Matrix plot of the amino acid sequence of the IBR gpG protein (441) against the amino acid sequence of the PRV gpX protein (498) (12). (FIG. 9B) Alignment of the conserved region between IBR gpG protein, PRV gpX protein, and HSV-2 gpG protein (699) (9). Note that IUPAC-IUB Biochemical Nomenclature Commission conventions are used.

FIG. 10 Western blot of proteins released into the medium of IBR and PRV infected cells, showing the absence of gpG in S-PRV-013, S-IBR-035, S-IBR-036, S-IBR-037, and S-IBR-038 but its presence in S-PRV-160 and wild type S-IBR-000. Lanes (A) 0.5 μg purified gpG, (B) blank lane, (C) S-PRV-160, (D) S-PRV-013, (E) pre-stained molecular weight markers, (F) 0.5 μg purified gpG, (G) S-IBR-038, (H) S-IBR-037, (I) S-IBR-036, (J) S-IBR-035, (K) S-IBR-000, (L) uninfected MDBK cells, (M) pre-stained molecular weight markers. Media samples were prepared as described in the PREPARATION OF HERPESVIRUS CELL LYSATES. The concentrated media from the infection of one 6 cm dish of infected cells was loaded in each sample lane except for samples S-PRV-013 AND S-PRV-160 for which the media from two 6 cm dishes were loaded.

FIG. 11 SEQ ID NOS: 20-25 Detailed description of the DNA insertion in Plasmid 459-12.6. Diagram showing the orientation of DNA fragments assembled in plasmid 459-12.6. The origin of each fragment is indicated in the table. The sequences (SEQ ID NOS: 20-25) located at each of the junctions between fragments is also shown. The restriction sites used to generate each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, !, indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique glycoprotein G (gpG), glycoprotein III (gpIII), glycoprotein X (gpX), polyadenylation signal (pA), infectious bovine rhinotracheitis virus (IBR), pseudorabies virus (PRV), and human cytomegalovirus (HCMV).

FIG. 12 SEQ ID NOS: 26-32 Detailed description of the DNA insertion in Homology Vector 439-01.31. Diagram showing the orientation of DNA fragments assembled in plasmid 439-01.31. The origin of each fragment is indicated in the table. The sequences (SEQ ID NOS: 26, 27 and 29-32) located at each of the junctions between fragments is also shown. The restriction sites used to generate each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, !, indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique short 2 (US2), glycoprotein G (gpG), glycoprotein IV (gpIV), polyadenylation signal (pA), infectious bovine rhinotracheitis virus (IBR), pseudorabies virus (PRV), and human cytomegalovirus (HCMV).

FIG. 13 SEQ ID NOS: 29-32, 33 and 34 Detailed description of the DNA insertion in Homology Vector 439-21.69. Diagram showing the orientation of DNA fragments assembled in plasmid 439-21.69. The origin of each fragment is indicated in the table. The sequences (SEQ ID NOS: 29-32, 33 and 34) located at each of the junctions between fragments is also shown. The restriction sites used to generate each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, !, indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique short 2 (US2), glycoprotein G (gpG), glycoprotein IV (gpIV), polyadenylation signal (pA), infectious bovine rhinotracheitis virus (IBR), pseudorabies virus (PRV), and human cytomegalovirus (HCMV).

FIG. 14 SEQ ID NOS: 32, 33 and 35 Detailed description of the DNA insertion in Homology Vector 439-70.4. Diagram showing the orientation of DNA fragments assembled in plasmid 439-70.4. The origin of each fragment is indicated in the table. The sequences (SEQ ID NOS: 32, 33 and 35) located at each of the junctions between fragments is also shown. The restriction sites used to generated each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, !, indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: glycoprotein G (gpG), glycoprotein IV (gpIV) infectious bovine rhinotracheitis virus (IBR).

FIG. 15 SEQ ID NO: 36 DNA sequence of the IBR glycoprotein E gene. The sequence of 2038 base pairs of the IBR unique short region, starting approximately 1325 base pairs upstream of the HindIII K/HindIII F junction in the HindIII K fragment, are shown. The glycoprotein E (gpE) gene is transcribed toward the HindIII K/HindIII F junction as indicated in FIG. 1. The translational start and termination of the gpE gene are indicated. Note that IUPAC-IUB Biochemical Nomenclature Commission conventions are used.

FIGS. 16A and 16B SEQ ID NOS: 37-40 Homology between the IBR gpE (SEQ ID NO: 40) protein and the gpE protein of HSV-1 (SEQ ID NO: 37), the gpI protein of VZV (SEQ ID NO: 39), and the gI protein of PRV (SEQ ID NO: 38). (FIG. 16A) Matrix plot of the amino acid sequence of the IBR gpE protein (617) against the amino acid sequence of the PRV gI protein (577) (64). (FIG. 16B) Alignment of the conserved region between IBR gpE protein, PRV gI protein, and VZV gpI protein (37).

FIG. 17 SEQ ID NOS: 41-43 Detailed description of a plasmid containing the gpE gene. Diagram showing the orientation of DNA fragments to be assembled in the gpE-containing plasmid. The origin of each fragment is indicated in the table. The sequences (SEQ ID NOS: 41-43) located at each of the junctions between fragments are also shown. The restriction sites used to generate each fragment are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, !, indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: unique glycoprotein E (gpE), glycoprotein IV (gIV), and infectious bovine rhinotracheitis virus (IBR).

FIG. 18 SEQ ID NOS: 44-48 Detailed description of the DNA insertion in the homology vector 536-03.5. Diagram showing the orientation of DNA fragments to be assembled in the homology vector. The origin of each fragment is indicated in the table. The sequences (SEQ ID NOS: 44-48) located at each of the junctions between fragments is also shown. The restriction sites used to generate each fragment as well as synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, ( ), refer to amino acids, and restriction sites in brackets, !, indicate the remnants of sites which were destroyed during construction. The following abbreviations are used: glycoprotein E (gpE), immediate early promoter (IE), infectious bovine rhinotracheitis virus (IBR), and pseudorabies virus (PRV).

FIG. 19 Construction of Recombinant S-IBR-004 Virus. S-IBR-004 is an IBR recombinant virus carrying an inserted foreign gene (NEO) under the control of the PRV gpX promoter. A new XbaI site was created at the short unique region and the original SacI site was deleted.

FIG. 20 Construction of Recombinant S-IBR-008 Virus. S-IBR-008 is a recombinant IBR virus that has a bovine rotavirus glycoprotein gene and the plasmid vector inserted at the XbaI site in the unique long region. A site specific deletion was created at the SacI! site due to the loss of NEO gene in the short unique region.

FIG. 21 SEQ ID NO: 49 Sequence of the PI-3 (SF-4 Strain) HN Gene. Note that IUPAC-IUB Biochemical Nomenclature Commission conventions are used.

FIGS. 22A-22C Details of S-IBR-018 Construction.

FIG. 22A First line shows the IBR (Cooper Strain) BamHI-C fragment map. Second line shows the construction of the alpha-4 promoter on the PI-3 HN gene and its insertion into the HindIII site in BamHI-C. Also shown are the beta-gal and neomycin (NEO) gene constructions under the control of the gX promoter that were put into the XbaI site and used as selectable markers to purify the recombinant virus.

FIG. 22B The BamHI-C fragment map of S-IBR-018 after insertion of the PI-3 HN, beta-gal, and neomycin genes.

FIG. 22C The S-IBR-018 genome showing the location of the three inserted foreign genes.

Legend: B=BamHI; H=HindIII; X=XbaI; S=StuI; UL=unique long region; US=unique short region; IR=internal repeat region; TR=terminal repeat region.

FIGS. 23A-23C Details of S-IBR-019 Construction.

FIG. 23A First line shows the IBR (Cooper Strain) BamHI-C fragment map. Second line shows the construction of the alpha-4 promoter on the PI-3 F gene and its insertion into the HindIII site in BamHI-C. Also shown are the beta-gal and neomycin (NEO) gene constructions under the control of the gX promoter that were put into the XbaI site and used as selectable markers to purify the recombinant virus.

FIG. 23B The BamHI-C fragment map of S-IBR-019 after insertion of the PI-3 F, beta gal, and neomycin genes.

FIG. 23C The S-IBR-019 genome showing the location of the three inserted foreign genes.

Legend: B=BamHI; H=HindIII; X=XbaI; S=StuI; UL=unique long region; US=unique short region; IR=internal repeat region; TR=terminal repeat region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein. The DNA encoding gpG glycoprotein may be deleted or foreign DNA may be inserted into the DNA encoding gpG glycoprotein. The DNA encoding gpG glycoprotein may be deleted and foreign DNA may be inserted in place of the deleted DNA encoding gpG glycoprotein.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein and no gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted or foreign DNA may be inserted into the DNA encoding gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted and foreign DNA may be inserted in place of the deleted DNA encoding gpE glycoprotein.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein, DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and DNA encoding the gpE glycoprotein has been altered or deleted.

The present invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and (2) DNA encoding gpG glycoprotein has been altered or deleted. The DNA encoding the gpG glycoprotein may be deleted or foreign DNA may be inserted in place of the deleted DNA encoding gpG glycoprotein. Foreign DNA may be inserted in place of the deleted DNA corresponding to the US2 region of the naturally-occurring IBR virus.

The present invention also provides S-IBR-037, a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and (2) DNA encoding gpG glycoprotein has been deleted. S-IBR-037 has been deposited on Apr. 16, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2320.

The present invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted and a foreign DNA sequence which encodes Escherichia coli β-galactosidase has been inserted in place of the deleted DNA encoding gpG glycoprotein, and (2) DNA encoding gpG glycoprotein has been altered or deleted. The present invention also provides two examples of such viruses, S-IBR-035 and S-IBR-036.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted or foreign DNA may be inserted in the DNA encoding gpE glycoprotein. The DNA encoding gpE glycoprotein may be deleted and foreign DNA may be inserted in place of the deleted DNA encoding gpE glycoprotein.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein and DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted. Foreign DNA may be inserted into the DNA of the recombinant IBR virus. The foreign DNA may be inserted into the XbaI site in the long unique region. The foreign DNA may be a sequence which encodes bovine rotavirus glycoprotein 38; this sequence may be inserted into the XbaI site in the long unique region.

The present invention provides S-IBR-008, a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted and in which a foreign DNA sequence which encodes bovine rotavirus glycoprotein 38 has been inserted into the XbaI site in the long unique region. S-IBR-008 has been deposited on Jun. 18, 1986 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2141.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted and (2) at least a portion of both repeat sequences has been deleted. The present invention further provides an example of such a recombinant virus, designated S-IBR-027. S-IBR-027 has been deposited on Apr. 17, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2322.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which at least a portion of both repeat sequences has been deleted.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted and (2) DNA encoding one or more EcoRV restriction sites has been deleted. The present invention further provides an example of such a recombinant virus, designated S-IBR-002. S-IBR-002 has been deposited Jun. 18, 1986 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2140.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted and (2) wherein foreign DNA has been inserted into the DNA of the recombinant IBR virus. The foreign DNA may be a sequence which encodes the Tn5 NEO gene.

The present invention further provides S-IBR-020, a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted and (2) wherein a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the DNA of the recombinant IBR virus.

The present invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted, (2) wherein a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the DNA of the recombinant IBR virus, and (3) wherein at least a portion of the thymidine kinase gene has been deleted.

The present invention also provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which (1) at least a portion of both repeat sequences has been deleted, (2) wherein a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the DNA of the recombinant IBR virus and the Tn5 NEO gene is under the control of an inserted, upstream, HSV-1 alpha-4 promoter, and (3) wherein at least a portion of the thymidine kinase gene has been deleted. The subject invention provides an example of such a recombinant virus, designated S-IBR-028. S-IBR-028 has been deposited on May 14, 1991 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2326.

The present invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the viral DNA. The Tn5 NEO gene may be under the control of an inserted, upstream, pseudorabies virus glycoprotein X promoter. The subject invention further provides an example of a recombinant virus wherein the Tn5 NEO gene is under the control of an inserted, upstream, pseudorabies virus glycoprotein X promoter, designated S-IBR-004. S-IBR-004 has been deposited on May 23, 1986 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2134.

The subject invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Escherichia coli β-galactosidase and Tn5 NEO genes, and the parainfluenzae-3 virus hemaglutinin gene, HN, has been inserted into the viral DNA. The subject invention provides an example of such a recombinant virus, designated S-IBR-018.

The subject invention further provides a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Escherichia coli β-galactosidase and Tn5 NEO genes, and the parainfluenzae-3 virus virus fusion gene, F, has been inserted into the viral DNA. The subject invention provides an example of such a recombinant virus, designated S-IBR-019.

The recombinant viruses of the subject invention were derived from the Cooper Strain. However, other IBR viruses, such as the LA strain or the 3156 strain, may also be used.

The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of any of the recombinant viruses of the present invention. The vaccine may contain either inactivated or live recombinant virus.

Suitable carriers for the recombinant virus are well known in the art and include proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as hydrolyzed proteins, lactose, etc. Preferably, the live vaccine is created by taking tissue culture fluids and adding stabilizing agents such as stabilized, hydrolyzed proteins. Preferably, the inactivated vaccine uses tissue culture fluids directly after inactivation of the virus.

The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein.

The subject invention provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein and no gpE glycoprotein.

The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein, DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and DNA encoding the gpE glycoprotein has been altered or deleted.

The subject invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and (2) DNA encoding gpG glycoprotein has been altered or deleted.

The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein.

The subject invention provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein and DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted.

The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted.

The subject invention provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus from which at least a portion of both repeat sequences has been deleted.

The subject invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Tn5 NEO gene has been inserted into the viral DNA.

The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Escherichia coli β-galactosidase and Tn5 NEO genes, and the parainfluenzae-3 virus hemaglutinin gene, HN, has been inserted into the viral DNA.

The subject invention also provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which a foreign DNA sequence which encodes the Escherichia coli β-galactosidase and Tn5 NEO genes, and the parainfluenzae-3 virus fusion gene, F, has been inserted into the viral DNA.

All of the vaccines described above may contain either inactivated or live recombinant virus. The vaccines may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneous, intraperitoneal, or intravenous injection. Alternatively, the vaccine may be administered intranasally or orally.

The present invention also provides a method of immunizing an animal against infectious bovine rhinotracheitis virus which comprises administering to the animal an effective immunizing dose of any of the vaccines of the present invention. The animal may be a bovine.

The subject invention also provides a method for distinguishing an animal vaccinated with a vaccine which comprises an effective immunizing amount of a recombinant virus of the present invention from an animal infected with a naturally-occurring IBR virus which comprises analyzing a sample of a body fluid from the animal for the presence of gpG glycoprotein of IBR virus and at least one other antigen normally expressed in an animal infected by a naturally-occurring IBR virus, identifying antigens which are present in the body fluid, and determining whether gpG glycoprotein is present in the body fluid. The presence of antigens which are normally expressed in an animal by a naturally-occurring IBR virus and the absence of gpG glycoprotein in the body fluid is indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus. The presence of antigens and gpG glycoprotein in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigens and gpG glycoprotein.

One of the vaccines that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein. Another vaccine that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein and no gpE glycoprotein. Yet another vaccine that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein, DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and DNA encoding the gpE glycoprotein has been altered or deleted. Still another vaccine that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which (1) DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and (2) DNA encoding gpG glycoprotein has been altered or deleted.

The present invention also provides a method for distinguishing an animal vaccinated with a vaccine which comprises an effective immunizing amount of a recombinant virus of the present invention from an animal infected with a naturally-occurring IBR virus which comprises analyzing a sample of a body fluid from the animal for the presence of gpE glycoprotein of IBR virus and at least one other antigen normally expressed in an animal infected by a naturally-occurring IBR virus, identifying antigens which are present in the body fluid and determining nether gpE glycoprotein is present in the body fluid. The presence of antigens which are normally expressed in an animal by a naturally-occurring IBR virus and the absence of gpE glycoprotein in the body fluid is indicative of an animal vaccinated with the vaccine and not infected with a naturally-occurring IBR virus. The presence of antigens and gpE glycoprotein in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigens and gpE glycoprotein.

One of the vaccines useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted and DNA encoding the gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein and no gpE glycoprotein. Another vaccine that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpG glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpG glycoprotein, DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted, and DNA encoding the gpE glycoprotein has been altered or deleted. Yet another vaccine that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein. Still another vaccine that is useful in this method is a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant IBR virus comprising viral DNA from a naturally-occurring IBR virus in which DNA encoding gpE glycoprotein has been altered or deleted so that upon replication the recombinant IBR virus produces no gpE glycoprotein and DNA corresponding to the US2 region of the naturally-occurring IBR virus has been deleted.

The present invention also provides isolated DNA encoding the gpG glycoprotein of IBR virus. The subject invention also provides purified recombinant gpG glycoprotein encoded by the DNA encoding the gpG glycoprotein of IBR virus. The subject invention further provides a recombinant cloning vector which comprises the DNA encoding the gpG glycoprotein of IBR virus. The subject invention also provides a recombinant expression vector which comprises the DNA encoding the gpG glycoprotein of IBR virus. The subject invention provides a host cell which comprises the recombinant expression vector which comprises the DNA encoding the gpG glycoprotein of IBR virus.

The subject invention also provides a method of producing a polypeptide which comprises growing the host cell which comprises the recombinant expression vector which comprises the DNA encoding the gpG glycoprotein of IBR virus under conditions such that the recombinant expression vector expresses gpG glycoprotein and recovering the gpG glycoprotein so expressed.

The subject invention also provides an antibody directed to an epitope of the purified gpG glycoprotein of IBR virus encoded by the DNA encoding the gpG glycoprotein of IBR virus. The antibody may be a monoclonal antibody.

The subject invention also provides a method of detecting the presence or absence of gpG glycoprotein of IBR virus in a sample which comprises contacting the sample with an antibody directed to an epitope of the purified gpG glycoprotein of IBR virus encoded by the DNA encoding the gpG glycoprotein of IBR virus under conditions such that the antibody forms a complex with any gpG glycoprotein present in the sample and detecting the presence or absence of such complex. The sample may be bovine-derived.

The subject invention also provides isolated DNA encoding the gpE glycoprotein of IBR virus. The subject invention also provides purified recombinant gpE glycoprotein encoded by the DNA encoding the gpE glycoprotein of IBR virus. The subject invention further provides a recombinant cloning vector which comprises the DNA encoding the gpE glycoprotein of IBR virus. The subject invention provides a recombinant expression vector which comprises the DNA encoding the gpE glycoprotein of IBR virus. The subject invention also provides a host cell which comprises the recombinant expression vector which comprises the DNA encoding the gpE glycoprotein of IBR virus.

The subject invention also provides a method of producing a polypeptide which comprises growing the host cell which comprises the recombinant expression vector which comprises the DNA encoding the gpE glycoprotein of IBR virus under conditions such that the recombinant expression vector expresses gpE glycoprotein and recovering the gpE glycoprotein so expressed.

The subject invention also provides an antibody directed to an epitope of the purified gpE glycoprotein of IBR virus encoded by the DNA encoding the gpE glycoprotein of IBR virus. The antibody may be a monoclonal antibody.

The subject invention also provides a method of detecting the presence or absence of gpE glycoprotein of IBR virus in a sample which comprises contacting the sample with an antibody directed to an epitope of the purified gpE glycoprotein of IBR virus encoded by the DNA encoding the gpE glycoprotein of IBR virus under conditions such that the antibody forms a complex with any gpE glycoprotein present in the sample and detecting the presence or absence of such complex. The sample may be bovine-derived.

The subject invention also provides a method of producing a fetal-safe, live recombinant IBR virus which comprises treating viral DNA from a naturally-occurring live IBR virus so as to delete from the virus DNA corresponding to the US2 region of the naturally-occurring IBR virus.

The subject invention also provides a recombinant pseudorabies virus designated S-PRV-160. The subject invention also provides an antibody directed to an epitope of the recombinant pseudorabies virus designated S-PRV-160.

Materials and Methods

PREPARATION OF IBR VIRUS STOCK SAMPLES. IBR virus stock samples were prepared by infecting MDBK cells at a multiplicity of infection of 0.01 PFU/cell in Dulbecco's Modified Eagle Medium (DMEM) containing 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components were obtained from Irvine Scientific or an equivalent supplier, and hereafter are referred to as complete DME medium) plus 1% fetal bovine serum. After cytopathic effect was complete, the medium and cells were harvested and the cells were pelleted at 3000 rpm for 5 minutes in a clinical centrifuge. Cells were resuspended in 1/10 the original volume of medium, and an equal volume of skim milk (9% skim milk powder in H₂ O weight/volume) was added. The virus sample was frozen at -70° C. The titers were usually about 10⁸ PFU/ml.

PREPARATION OF HERPESVIRUS DNA. For herpesvirus DNA preparation, a confluent monolayer of cells (MDBK for IBR virus or Vero for PRV) in a 25 cm² flask or 60 mm petri dish was infected with 100 μl of virus sample. After overnight incubation, or when the cells were showing 100% cytopathic effect, the cells were scraped into the medium. The cells and medium were centrifuged at 3000 rpm for 5 minutes in a clinical centrifuge. The medium was decanted, and the cell pellet was gently resuspended in 0.5 ml of solution containing 0.5% Nonident P-40 (NP-40, purchased from Sigma Chemical Co., St. Louis, Mo.). The sample was incubated at room temperature for 10 minutes. Ten μl of a stock solution of RNase A (Sigma) was added (stock was 10 mg/ml, boiled for 10 minutes to inactivate DNAse). The sample was centrifuged to pellet nuclei. The DNA pellet was removed with a pasteur pipette or wooden stick and discarded. The supernatant fluid was decanted into a 1.5 ml Eppendorf tube containing 25 μl of 20% sodium dodecyl sulfate (Sigma) and 25 μl proteinase-K (10 mg/ml; Boehringer Mannheim). The sample was mixed and incubated at 37° C. for 30-60 minutes. An equal volume of water-saturated phenol was added and the sample was mixed briefly. The sample was centrifuged in an Eppendorf minifuge for 5 minutes at full speed. The upper aqueous phase was removed to a new Eppendorf tube, and two volumes of absolute ethanol were added and the tube put at -20° C. for 30 minutes to precipitate nucleic acid. The sample was centrifuged in an Eppendorf minifuge for 5 minutes. The supernatant was decanted and the pellet was washed with ⁻ 300 μl of 80% ethanol, followed by centrifugation in an Eppendorf minifuge for 5 minutes. The supernatant was decanted, and the pellet was air dried and rehydrated in ⁻ 16 μl H₂ O. For the preparation of larger amounts of DNA, the procedure was scaled up to start with a 850 cm² roller bottle of MDBK cells. The DNA was stored in 0.01M tris pH 7.5, 1 mM EDTA at 4° C.

PREPARATION OF HERPESVIRUS CELL LYSATES. For cell lysate preparation, serum free medium was used. A confluent monolayer of cells (MDBK for IBR virus or Vero for PRV) in a 25 cm² flask or a 60 mm petri dish was infected with 100 μl of virus sample. After cytopathic effect was complete, the medium and cells were harvested and the cells were pelleted at 3000 rpm for 5 minutes in a clinical centrifuge. For media samples medium was concentrated approximately 10-fold by filtration with a centricon-10 microconcentrator (Amicon). For cell samples the cell pellet was resuspended in 250 μl of disruption buffer (2% sodium dodecyl sulfate, 2% β-mercaptoethanol). The samples were sonicated for 30 seconds on ice and stored at -20° C.

WESTERN BLOTTING PROCEDURE. Samples of lysates, controls and protein standards were run on a polyacrylamide gel according to the procedure of Laemmli (2). After gel electrophoresis the proteins were transferred according to Sambrook (14). The primary antibody was a mouse hyper-immune serum raised against chemically-synthesized gpG peptides (amino acids 232-252 and 267-287) linked to keyhole limpet hemocyanin. The secondary antibody was a goat anti-mouse alkaline phosphatase coupled antibody.

MOLECULAR BIOLOGICAL TECHNIQUES. Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels, ligation, phosphorylation with kinase, treatment with phosphatase, growth of bacterial cultures, transformation of bacteria with DNA, and other molecular biological methods are described by Maniatis (6). Except as noted, these were used with minor variation.

LIGATION. DNA was joined together by the action of the enzyme T4 DNA ligase (BRL). Ligation reactions contained various amounts of DNA (from 0.2 to 20 μg), 20 mM Tris pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol (DTT), 200 μM ATP and 20 units T4 DNA ligase in 10-20 μl final reaction volume. The ligation proceeded for 3-16 hours at 15° C.

DNA SEQUENCING. Sequencing was performed using the BRL "SEQUENASE", a modified bacteriophage T7 DNA polymerase (United States Biochemicals, Cleveland, Ohio) Kit and ³⁵ S-dATP (NEN). Reactions using both the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, compressed areas were resolved on formamide gels. Templates were double-stranded plasmid subclones or single stranded M13 subclones, and primers were either made to the vector just outside the insert to be sequenced, or to previously obtained sequence. Sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with Superclone and Supersee programs from Coral Software.

SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis (6). DNA was blotted to nitrocellulose filters and hybridized to appropriately labeled DNA probes. Probes for southern blots were prepared using either the Nonradioactive DNA Labeling and Detection Kit of Boehringer Mannheim or the nick translation kit of Bethesda Research Laboratories (BRL). In both cases the manufacturers' recommended procedures were followed.

DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS. The method is based upon the calcium phosphate procedure of Graham and Van der Eb (4) with the following modifications. Virus and/or plasmid DNA were diluted to 298 μl in 0.01M Tris pH 7.5, 1 mM EDTA. Forty μl 2M CaCl₂ was added followed by an equal volume of 2×HEPES buffered saline (10 g N-2-hydroxyethyl piperazine N'-2-ethanesulfonic acid (HEPES), 16 g NaCl, 0.74 g KCl 0.25 g Na₂ HPO₄ u2H₂ O, 2 g dextrose per liter H₂ O and buffered with NaOH to pH 7.4). The mixture was then incubated on ice for 10 minutes, and then added dropwise to an 80% confluent monolayer of MDBK or rabbit skin (RS) cells growing in a 60 mm petri dish under 5 ml of medium (DME plus 2% fetal bovine serum). The cells were incubated 4 hours at 37° C. in a humidified incubator containing 5% CO₂. The cells were then washed with three 5 ml aliquots of 1×PBS (1.15 g Na₂ HPO₄, 0.2 g KH₂ PO₄, 0.8 g NaCl, 0.2 g KCl per liter H₂ O), and fed with 5 ml of medium (DME plus 2% fetal bovine serum). The cells were incubated at 37° C. as above for 3-7 days until cytopathic effect from the virus was 50-100%. Virus was harvested as described above for the preparation of virus stocks. This stock was referred to as a transfection stock and was subsequently screened for recombinant.virus by the BLUOGAL SCREEN FOR RECOMBINANT IBR VIRUS.

HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. This method relies upon the homologous recombination between herpesvirus DNA and plasmid homology vector DNA which occurs in tissue culture cells co-transfected with these elements. From 0.1-1.0 μg of plasmid DNA containing foreign DNA flanked by appropriate herpesvirus cloned sequences (the homology vector) were mixed with approximately 0.3 μg of intact herpesvirus DNA. The DNAs were diluted to 298 μl in 0.01M Tris pH 7.5, 1 mM EDTA and transfected into MDBK cells according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS (see above).

DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Rather than using homology vectors and relying upon homologous recombination to generate recombinant virus, we have also developed the technique of direct ligation to engineer herpesviruses. In this instance, a cloned foreign gene did not require flanking herpesvirus DNA sequences but only required that it have restriction sites available to cut out the foreign gene fragment from the plasmid vector. A compatible restriction enzyme was used to cut herpesvirus DNA. A requirement of the technique was that the restriction enzyme used to cut the herpesvirus DNA must cut at a limited number of sites. We have used XbaI, which cuts IBR virus DNA in one place. We have also used EcoRV which cuts IBR virus DNA in two places. For PRV we have used XbaI and HindIII, both of which cut in two places. Restriction sites previously introduced into herpesviruses by other methods may also be used. The herpesvirus DNA was mixed with a 30-fold molar excess of plasmid DNA (typically 5 μg of virus DNA to 10 μg of plasmid DNA), and the mixture was cut with the appropriate restriction enzyme. The DNA mixture was phenol extracted and ethanol precipitated to remove restriction enzymes, and ligated together according to the ligation procedure detailed above. The ligated DNA mixture was then resuspended in 298 μl 0.01M Tris pH 7.5, 1 mM EDTA and transfected into cells (MDBK or RS for IBR virus and Vero for PRV) according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS (see above). The direct ligation procedure may also be used to delete DNA from herpesviruses. Non-essential DNA which is flanked by appropriate restriction enzyme sites may be deleted by digesting the virus DNA with such enzymes and religation. The frequency of engineered viruses generated by the direct ligation procedure is high enough that screening can be accomplished by restriction enzyme analysis of randomly picked plaques from the transfection stock.

BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. When the E.coli β-galactosidase (lacZ) marker gene was incorporated into a recombinant virus the plaques containing recombinants were visualized by a simple assay. The chemical Bluogal™ (Bethesda Research Labs) was incorporated (200 μg/ml) into the agarose overlay during the plaque assay, and plaques that expressed active β-galactosidase turned blue. The blue plaques were then picked onto fresh cells (MDBK for IBR virus and Vero for PRV) and purified by further blue plaque isolations. In recombinant virus strategies in which the E.coli β-galactosidase marker gene is removed, the assay involves plaque purifying white plaques from a background of parental blue plaques. In both cases viruses were typically purified with three rounds of plaque purification.

ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS. A third method for screening the recombinant virus stock was to look directly for the expression of the foreign gene with antibodies. Herpesvirus plaques were spotted and picked by inserting a toothpick through the agarose above the plaque and scraping the plaque area on the dish. Viruses were then rinsed from the toothpick by inserting the toothpick into a well of a 96-well micro-titer dish (Falcon Plastics) containing a confluent monolayer of tissue culture cells that had been washed 3 times in DME medium without serum. It was important for the virus to grow without serum at this stage to allow the immunological procedure to work. After cytopathic effect was complete, the plates were put at -70° C. to freeze and lyse the cells. The medium was thawed, and the freeze/thaw procedure was repeated a second time. Then 50-100 microliters of medium were removed from each well and filtered under vacuum through a nitrocellulose membrane (S&S BA85) using a DotBlot˜ apparatus (BRL). The filter blots were soaked in a blocking solution of 0.01M Tris pH 7.5, 0.1M NaCl, 3% bovine serum albumin at room temperature for two hours with shaking. The filter blots were then placed in a sealable bag (Sears Seal-A-Meal™ or equivalent), and 10 mls of the blocking solution that contained 10 microliters of antibody specific for the foreign protein were added. After overnight incubation at room temperature with shaking, the blot was washed 3 times with 100 mls 0.01M Tris, pH 7.5, 0.1M NaCl, 0.05% Tween 20 detergent (Sigma). The blot was put in another sealable bag and 10 mls blocking solution containing 10⁶ counts per minute of ¹²⁵ I-protein A (New England Nuclear) were added. After allowing the protein A to bind to the antibody for 2 hours at room temperature with shaking, the blot was washed as above, dried, and overlayed with an X-ray film and an intensifying screen (Dupont) and autoradiographed for 1-3 days at -70° C. The film was developed by standard procedures. Virus from the positive wells which contained the recombinant virus was further purified.

SELECTION OF G418 RESISTANT IBR VIRUS. The antibiotic G418 (GIBCO) has a wide range of inhibitory activity on protein synthesis. However, recombinant viruses expressing the aminoglycosidase 3'-phosphotransferase, encoded by the NEO gene of the transposable element Tn5, are resistant to G418. The transfection stocks of recombinant viruses were grown on MDBK cells in the presence of 500 μg/ml G418 in complete DME medium plus 1% fetal bovine serum. After one or two days at 37° C., plaques from the dishes inoculated with the highest dilution of virus were picked for virus stocks. The selection was repeated a second or third time. The virus stocks generated from the G418 selection were tested for NEO gene insertion by the SOUTHERN BLOTTING OF DNA hybridization procedure described above.

CONSTRUCTION OF DELETION VIRUSES. The strategy used to construct deletion viruses involved the use of both homologous recombination and direct ligation techniques. Initially a virus was constructed via homologous recombination, in which the gene to be deleted was replaced with the E.coli β-galactosidase marker gene. A second virus was then constructed in which the marker gene was deleted either by homologous recombination or via direct ligation. The advantage of this strategy is that both viruses may be purified by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. The first virus is purified by picking blue plaques from a white plaque background, the second virus is purified by picking white plaques from a blue plaque background. Several homology vectors were constructed for the purpose of deleting the gpG and gpE gene coding regions. A detailed description of these homology vectors follows.

HOMOLOGY VECTOR 129-71.5. The plasmid 129-71.5 was constructed for the purpose of deleting a portion of the TK gene coding region from the IBR virus. It incorporates a selectable marker, the bacterial transposon neomycin resistance gene, flanked by IBR virus DNA. Upstream of the marker gene is an approximately 860 base pair fragment of IBR virus DNA which ends with sequences encoding amino acids 1-62 of the TK primary translation product. Downstream of the marker gene is an approximately 1741 base pair fragment of IBR virus DNA which begins with sequences encoding amino acids 156-367 of the TK primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding for amino acids 63-155 of the TK primary translation product with DNA coding for the marker gene. Note that the marker gene will be under the control of the herpes simplex type 1 alpha-4 immediate early gene promoter (5). A detailed description of the plasmid is given in FIG. 7. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6). It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 7. The plasmid vector is derived from an approximately 2975 base pair SmaI to HindIII restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 860 base pair NcoI to BamHI restriction fragment of the IBR virus HindIII restriction fragment A (7). This fragment is located on an approximately 5500 base pair ClaI to NruI fragment contained in the IBR virus HindIII A fragment. Fragment 2 is an approximately 490 base pair PvuII to BamHI restriction sub-fragment of the HSV-1 BamHI restriction fragment N (5). Note that the HSV-1 oriS region has been removed from this fragment by deletion of the sequences between the SmaI sites located 1483 and 128 base pairs away from the PvuII end (10) Fragment 3 is an approximately 1541 base pair BglII to BamHI restriction fragment of plasmid pNEO (P.L. Biochemicals, Inc.). Fragment 4 is an approximately 784 base pair SmaI to SmaI restriction sub-fragment of the HSV-1 BamHI restriction fragment Q (10). Note that this fragment is oriented such that the polyadenylation sequence (AATAAA) is located closest to junction D. Fragment 5 is an approximately 1741 base pair BglII to StuI restriction sub-fragment from the IBR HindIII restriction fragment A (7).

PLASMID 459-12.6. The plasmid 459-12.6 was generated for the purpose of constructing a recombinant cloning vector which expresses the IBR virus glycoprotein G. This was accomplished by inserting the IBR virus gpG gene into S-PRV-013 (U.S. Ser. No. 06/823,102, filed Jan. 27, 1986). Plasmid 459-12.6 contains a chimeric gene under the control of the IBR virus gpG promoter. The chimeric gene expresses a fusion protein consisting of the first 362 amino acids of IBR virus gpG fused to amino acids 421-467 of the PRV gpIII (13) followed by amino acids 480-498 of the PRV gpX (12). The chimeric gene is flanked by HindIII restriction sites. When this plasmid is used with S-PRV-013 and the restriction enzyme HindIII according to the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS the resulting recombinant will express the IBR virus gpG. A detailed description of the plasmid is given in FIG. 11. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6). It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 11. The plasmid vector is derived from an approximately 2999 base pair XbaI to XbaI restriction fragment of a hybrid cloning vector derived from pSP64 and pSP65 (Promega). The hybrid cloning vector was constructed by joining the approximately 1369 base pair PvuI to SmaI fragment from pSP64 with the approximately 1652 base pair PvuI to SmaI fragment from pSP65. Fragment 1 is an approximately 182 base pair PstI to EcoRV restriction sub-fragment of the HCMV XbaI restriction fragment B (16). Fragment 2 is an approximately 2121 base pair MluI to XhoI restriction sub-fragment of the IBR virus HindIII restriction fragment K (7). Fragment 3 is an approximately 121 base pair XhoI to BamHI restriction sub-fragment of the PRV BamHI restriction fragment #2 (3). Fragment 4 is an approximately 760 base pair NdeI to SalI restriction sub-fragment of the PRV BamHI restriction fragment #7 (3).

HOMOLOGY VECTOR 439-01.31. The plasmid 439-01.31 was constructed for the purpose of deleting a portion of the gpG gene coding region from the IBR virus. It incorporates an E.coli β-galactosidase marker gene flanked by IBR virus DNA. Downstream of the marker gene is an approximately 3593 base pair fragment of IBR virus DNA which ends with sequences encoding the first 262 amino acids of the gpG primary translation product. Upstream of the marker gene is an approximately 785 base pair fragment of IBR virus DNA which begins with sequences encoding the last 80 amino acids of the gpG primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS it will replace the DNA coding for amino acids 263-361 of the gpG primary translation product with DNA coding for the marker gene. Note that the β-galactosidase (lacZ) marker gene will be under the control of the human cytomegalovirus immediate early gene promoter. A detailed description of the plasmid is given in FIG. 12. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6). It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 12. The plasmid vector is derived from an approximately 2965 base pair HindIII to SmaI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 3593 base pair HindIII to XhoI restriction fragment of the IBR HindIII restriction fragment K (7). Fragment 2 is an approximately 753 base pair SalI to NdeI restriction fragment of the PRV BamHI restriction fragment #7 (3). Note that this fragment was resected with Exonuclease III/S1 nuclease digestion such that approximately 57 base pairs were removed from the NdeI end. Fragment 3 is an approximately 3347 base pair BalI to BamHI restriction fragment of plasmid pJF751 (38). Fragment 4 is an approximately 1191 base pair AvaI to PstI restriction fragment from the HCMV XbaI restriction fragment E (16). Fragment 5 is an approximately 785 base pair XhoI to NdeI restriction fragment from the IBR HindIII restriction fragment K (7). Note that the lacZ marker gene is flanked by XbaI sites located at Junction B and Junction E in this plasmid permiting the marker gene to be cut out with XbaI.

HOMOLOGY VECTOR 439-21.69. The plasmid 439-21.69 was constructed for the purpose of deleting a portion of the gpG gene coding region from the IBR virus. It incorporates an E.coli β-galactosidase (lacZ) marker gene flanked by IBR virus DNA. Downstream of the marker gene is an approximately 888 base pair fragment of IBR virus DNA which begins approximately 1042 base pairs upstream of the initiation codon of the gpG gene and ends approximately 154 base pairs upstream of the initiation codon of the gpG gene. Upstream of the marker gene is an approximately 785 base pair fragment of IBR virus DNA which begins with sequences encoding the last 80 amino acids of the gpG primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS it will replace the DNA coding for amino acids 1-361 of the gpG primary translation product with DNA coding for the marker gene. Note that the β-galactosidase (lacZ) marker gene will be under the control of the human cytomegalovirus immediate early gene promoter. A detailed description of the plasmid is given in FIG. 13. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6). It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 13. The plasmid vector is derived from an approximately 2965 base pair HindIII to SmaI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 3593 base pair HindIII to XhoI restriction fragment of the IBR HindIII restriction fragment K (7). Fragment 2 is an approximately 753 base pair SalI to NdeI restriction fragment of the PRV BamHI restriction fragment #7 (3). Note that this fragment was resected with Exonuclease III/S1 nuclease digestion such that approximately 57 base pairs were removed from the NdeI end. Fragment 3 is an approximately 3347 base pair BalI to BamHI restriction fragment of plasmid pJF751 (38). Fragment 4 is an approximately 1191 base pair AvaI to PstI restriction fragment from the HCMV XbaI restriction fragment E (16). Fragment 5 is an approximately 785 base pair XhoI to NdeI restriction fragment from the IBR HindIII restriction fragment K (7). Note that the lacZ marker gene is flanked by XbaI sites located at Junction B and Junction E in this plasmid permiting the marker gene to be cut out with XbaI.

HOMOLOGY VECTOR 439-70.4. The plasmid 439-70.4 was constructed for the purpose of deleting the E.coli β-galactosidase (lacZ) marker gene from S-IBR-035 virus. It incorporates two regions of IBR virus DNA which flanks the marker gene in S-IBR-035. The first region is an approximately 888 base pair fragment of IBR virus DNA which begins approximately 1042 base pairs upstream of the initiation codon of the gpG gene and ends approximately 154 base pairs upstream of the initiation codon of the gpG gene. The second region is an approximately 785 base pair fragment of IBR virus DNA which begins with sequences encoding the last 80 amino acids of the gpG primary translation product. When this plasmid is used in conjunction with S-IBR-035 DNA according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS it will delete the DNA coding for the E.coli β-galactosidase (lacZ) marker gene. A detailed description of the plasmid is given in FIG. 14. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (6). It may be constructed by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 14. The plasmid vector is derived from an approximately 2965 base pair HindIII to SmaI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 3593 base pair HindIII to XhoI restriction fragment of the IBR HindIII restriction fragment K (7). Fragment 2 is an approximately 785 base pair XhoI to NdeI restriction fragment from the IBR HindIII restriction fragment K (7).

IBR VIRUS gpE PLASMID. A plasmid may be generated for the purpose of constructing a recombinant cloning vector which expresses the IBR virus glycoprotein E. This plasmid may be used to insert the IBR virus gpE gene into S-PRV-002 (U.S. Pat. No. 4,877,737). The plasmid will contain the gpE gene flanked by XbaI restriction sites. When this plasmid is used with S-PRV-002 and the restriction enzyme XbaI according to the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS the resulting recombinant will express the IBR virus gpE. A detailed description of the plasmid is given in FIG. 17. It may be constructed, utilizing standard recombinant DNA techniques (6), by joining restriction fragments from the following sources. The plasmid vector is derived from an approximately 2999 base pair XbaI to XbaI restriction fragment of a hybrid cloning vector derived from pSP64 and pSP65 (Promega). The hybrid cloning vector was constructed by joining an approximately 1369 base pair PvuI to SmaI fragment from pSP64 with the approximately 1652 base pair PvuI to SmaI fragment from pSP65. Fragment 1 is an approximately 3647 base pair NdeI to HindIII restriction sub-fragment of the IBR virus HindIII restriction fragment K (7). Fragment 2 is an approximately 832 base pair HindIII to SacI restriction sub-fragment of an IBR virus 2400 base pair SmaI restriction fragment. This SmaI fragment has been cloned into the SmaI site of the plasmid pSP64 (Promega). This plasmid is designated PSY1645. PSY1645 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. 68650. Note that the lacZ marker gene is flanked by XbaI sites located at Junction B and Junction E in this plasmid permitting the marker gene to be cut out with XbaI.

HOMOLOGY VECTOR 536-03.5. The plasmid 536-03.5 was constructed for the purpose of deleting a portion of the gpE gene coding region from the IBR virus. It incorporates an E.coli β-galactosidase (lacZ) marker gene flanked by IBR virus DNA. Upstream of the marker gene is an approximately 1704 base pair fragment of IBR virus DNA which ends with sequences encoding amino acids 1-76 of the gpE primary translation product. Downstream of the marker gene is an approximately 742 base pair fragment of IBR virus DNA which begins with sequences encoding amino acids 548-617 of the gpE primary translation product. When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding for amino acids 77-547 of the gpE primary translation product with DNA coding for the marker gene. Note that the β-galactosidase (lacZ) marker gene will be under the control of the PRV gpX. A detailed description of the plasmid is given in FIG. 18. It may be constructed utilizing standard recombinant DNA techniques (6), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIG. 18. The plasmid vector is derived from an approximately 2975 base pair SmaI to HindIII restriction fragment of pSP65 (Promega). Fragment 1 is an approximately 1704 base pair SmaI to SmaI restriction sub-fragment of the IBR HindIII restriction fragment K (7). Fragment 2 is an approximately 413 base pair SalI to BamHI restriction sub-fragment of the PRV BamHI restriction fragment 10 (3). Fragment 3 is an approximately 3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751 (38). Fragment 4 is an approximately 754 base pair NdeI to SalI restriction sub-fragment of the PRV BamHI restriction fragment #7 (3). Fragment 5 is an approximately 742 base pair NheI to BglI sub-fragment of an IBR virus 2400 base pair SmaI fragment. This SmaI fragment has been cloned into the SmaI site of the plasmid pSP64 (Promega). This plasmid is designated PSY1645. PSY1645 has been deposited with the American Type Culture Collection. Note that the lacZ marker gene is flanked by XbaI sites located at Junction B and Junction E in this plasmid permitting the marker gene to be cut out with XbaI.

VACCINATION STUDIES IN CALVES WITH INACTIVATED IBR VIRUS. Calves, seronegative to IBR virus, were housed in facilities secure from IBR virus exposure. Groups of four calves were vaccinated intramuscularly with vaccines containing 10⁷.3 or 10⁸.0 plaque forming units of inactivated IBR virus formulated with an oil adjuvant. A second vaccination was given 21 days later; four calves were maintained as unvaccinated controls. At 21 days after the second vaccination, animals were challenged intranasally with virulent wild-type IBR virus. After vaccination and challenge, animals were observed and the injection site was palpated weekly. Blood samples were taken on days 0, 7, 21, 28, and 42 post vaccination. After challenge, animals were observed daily for clinical signs of IBR. Blood samples were taken on days 7 and 13 post challenge. Nasal swabs were collected on days 3, 6, 9, and 12 post challenge.

PURIFICATION OF IBR VIRUS gpG. gpG was purified from the tissue culture medium of infected MDBK cells. Confluent MDBK cells in serum-free medium were infected at a multiplicity of infection equal to 5, with wild-type, Cooper strain of IBR virus. The cells and media were harvested at approximately twenty-two hours after infection, when the cells showed considerable cytopathic effect and the fluids were centrifuged at 5000 rpm for 15 minutes.

The supernatant fluid was concentrated approximately 10-fold by ultrafiltration through an Amicon ym-30 membrane, and dialyzed against 10 mM NaPO₄ pH 7.2. The dialysate was treated for 20 minutes at 0° C. with 70% perchloric acid to a final concentration of 0.2M perchloric acid, then centrifuged at 12,000 rpm for 20 minutes. The supernatant fluid was then dialyzed against 20 mM Tris pH 9.5.

The acid-soluble proteins were separated by column chromatography on a DEAE-Sephacel anion exchange column using a liner gradient elution: 0 to 100% A to B where A=20 mM Tris pH 9.5 and B=20 mM Tris pH 9.5/800 mM NaCl. The gpG eluted at approximately 35-40% B. Peak fractions were assayed by Western blot using anti gpG peptide sera. Reactive fractions were combined and dialyzed against 5 mM Tris pH 7.0. The sample was then concentrated 10-fold by lyophilization and stored at -20° C.

ELISA ASSAY. A standard enzyme-linked immunosorbent assay (ELISA) protocol was used to determine the immune status of cattle following vaccination and challenge.

A purified gpG antigen solution (100 μl at 1 ng/μl in PBS) was allowed to absorb to the wells of microtiter dishes for 18 hours at 4° C. The coated wells were rinsed one time with PBS. Wells were blocked by adding 250 μl of PBS containing 1% BSA (Sigma) and incubating 1 hour at 37° C. The blocked wells were rinsed one time with PBS containing 0.02% Tween 20. 50 μl of test serum (previously diluted 1:2 in PBS containing 1% BSA) were added to the wells and incubated 1 hour at 37° C. The antiserum was removed and the wells were washed 3 times with PBS containing 0.02% Tween 20. 50 μl of a solution containing anti-bovine IgG coupled to horseradish peroxidase (diluted 1:500 in PBS containing 1% BSA, Kirkegaard and Perry Laboratories, Inc.) was added to visualize the wells containing antibody against the specific antigen. The solution was incubated 1 hour at 37° C., then removed and the wells were washed 3 times with PBS containing 0.02% Tween 20. 100 μl of substrate solution (ATBS, Kirkegaard and Perry Laboratories, Inc.) were added to each well and color was allowed to develop for 15 minutes. The reaction was terminated by addition of 0.1M oxalic acid. The color was read at absorbance 410 nm on an automatic plate reader.

PROCEDURE FOR GENERATING MONOCLONAL ANTIBODIES. To produce monoclonal antibodies, 8 to 10 week old BALB/c female mice were vaccinated intraperitoneally seven times at two to four week intervals with 10⁷ PFU of S-PRV-160. Three weeks after the last vaccination, mice were injected intraperitoneally with 40 μg of purified gpG. Spleens were removed from the mice three days after the last antigen dose.

Splenocytes were fused with mouse NS1/Ag4 plasmacytoma cells by the procedure modified from Oi and Herzenberg (39). Splenocytes and plasmacytoma cells were pelleted together by centrifugation at 300×g for 10 minutes. One ml of a 50% solution of polyethylene glycol (m.w. 1300-1600) was added to the cell pellet with stirring over one minute. Dulbecco's modified Eagles's medium (5 ml) was added to the cells over three minutes. Cells were pelleted by centrifugation at 300×g for 10 minutes and resuspended in medium with 10% fetal bovine serum and containing 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine (HAT). Cells (100 μl) were added to the wells of eight to ten 96-well tissue culture plates containing 100 μl of normal spleen feeder layer cells and incubated at 37° C. Cells were fed with fresh HAT medium every three to four days.

Hybridoma culture supernatants were tested by the ELISA ASSAY in 96-well microtiter plates coated with 100 ng of purified gpG. Supernatants from reactive hybridomas were further analyzed by black-plaque assay and by Western Blot. Selected hybridomas were cloned twice by limiting dilution. Ascitic fluid was produced by intraperitoneal injection of 5×10⁶ hybridoma cells into pristane-treated BALB/c mice.

METHOD FOR cDNA CLONING BOVINE ROTAVIRUS gp38 GENE. The Calf Nebraska strain of bovine rotavirus (USDA) was propagated on MA-104 cells (Rhesus monkey kidney cells from MA Bioproducts). Confluent monolayers were infected at a multiplicity of infection of greater than 10 in DMEM containing 5 micrograms/ml trypsin. Cells were incubated with virus for 48 hours or until a cytopathic effect was obtained. Media and cell debris were collected and centrifuged at 10,000×g for 20 minutes at 4° C. The supernatant containing the rotavirus was then centrifuged at 10,000×g in a preparative Beckman Ti45 rotor at 4° C. Virus pellets were resuspended in SM medium (50 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl₂) and homogenized lightly in a Dounce-type homogenizer. The resuspended virus was centrifuged at 10,000×g for 10 minutes then loaded onto 25-50% CsCl gradients in SM buffer. Gradients were centrifuged at 100,000×g for 4 hours at 20° C. The two blue-white bands representing intact virions and cores of rotavirus were collected, diluted, and the CsCl gradient procedure was repeated a second time. Virus obtained from the second gradient was dialyzed overnight against SM buffer at 4° C.

Dialyzed bovine rotavirus was twice extracted with an equal volume of SDS/phenol, then twice more with chloroform:isoamylalcohol (24:1). The double stranded RNA was precipitated with ethanol in the presence of 0.2M sodium acetate, centrifuged and resuspended in water. The yield was typically 100 micrograms from 1,000 cm² of infected cells.

160 micrograms of double-stranded bovine rotavirus RNA obtained from the above procedure was mixed with one microgram each of two synthetic oligonucleotide primers in a volume of 160 microliter (sequences of primers were: 5'-GGGAATTCTGCAGGTCACATCATACAATTCTAATCTAAG-3' (SEQ ID NO: 50) and 5'-GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCCGTTTGGCTA-3' (SEQ ID NO: 51)) derived from the published sequence of bovine rotavirus (40). The RNA-primer mixture was boiled for 3 minutes in a water bath then chilled on ice. Additions of 25 microliters of 1M Tris-HCl pH 8.3, 35 microliters of 1M KCl, 10 microliters of 0.25M MgCl₂, 7 microliters of 0.7M 2-mercaptoethanol, 7 microliters of 20 mM dNTP's, and 6 microliters of reverse transcriptase (100 units) were made sequentially. The reaction was incubated at 42° C. for 1.5 hours then 10 microliters of 0.5M EDTA pH 8.0 was added and the solution was extracted once with chloroform:phenol (1:1). The aqueous layer was removed and to it 250 microliters of 4M ammonium acetate and 1.0 ml of 95% ethanol was added, the mixture was frozen in dry ice and centrifuged in the cold. The resulting pellet was resuspended in 100 microliters of 10 mM Tris-HCl pH 7.5 and the ammonium acetate precipitation procedure was repeated. The pellet was resuspended in 100 microliters of 0.3M KOH and incubated at room temperature overnight, then at 37° C. for 2 hours. The solution was brought to neutral pH by addition of 10 microliters of 3.0M HCl and 25 microliters of 1.0M Tris-HCl pH 7.5. The resulting single-stranded cDNA was then precipitated two times by the above-described ammonium acetate-ethanol procedure. The pellet obtained was resuspended in 50 microliters of 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, boiled in a water bath for 2 minutes, then incubated at 59° C. for 16 hours. The solution was lyophilized to a volume of 15 microliters and the resulting double-stranded cDNA was run on a 1.0% agarose gel (Sigma agarose Type II). The ethidium bromide-stained DNA migrating at 1,000-1,100 base pair length was excised from the gel and electroeluted in a CBS electroeluter device. The solution was lyophilized, and the cDNA was resuspended in 25 microliters of water. To this solution was added 2 microliters of 1.0M Tris-HCl pH 7.5, 2 microliters of 1M KCl, 1 microliter of 0.25M MgCl₂, 1 microliter of 20 mM dNTP's and 5 units of E. coli DNA polymerase I. The reaction was incubated at room temperature for 15 minutes, then chloroform/phenol extracted and ammonium acetate-ethanol precipitated as described above. The resulting cDNA was tailed with dCTP using terminal deoxynucleotide transferase (BRL buffer and enzyme used). The reaction was stopped with 2 microliters of 0.5M EDTA, chloroform/phenol extracted and precipitated with sodium acetate in the presence of 10 micrograms of carrier tRNA. The resuspended cDNA was mixed with 200 ng of dGMP-tailed Pst I cut pBR322 (BRL catalog #5355SA) in 200 microliters of 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, heated to 65° C. for 5 minutes, then 57° C. for 2 hours. The annealed cDNA-vector pBR322 was transformed onto E. coli DH-1 cells prepared for high efficiency transformation. Colonies that showed sensitivity to ampicillin and tetracycline resistance were grown and DNA was prepared and cut with Pst I to determine the size of the cDNA insert. Several clones having Pst I inserts of 1,050-1,100 base pairs were analyzed and found to have identical restriction enzyme digest patterns. For one of these clones, the 1,100 base pair Pst I insert was subcloned into a M13 phage sequencing vector. Part of the DNA sequence of this clone was determined and was found to be identical to the published sequence (40).

cDNA CLONING. cDNA cloning refers to the methods used to convert RNA molecules into DNA molecules following state of the art procedures. Applicants' methods are described in Gubler and Hoffman (23). Bethesda Research Laboratories (Gaithersburg, Md.) have designed a cDNA Cloning Kit that is very similar to the procedures used by applicants and contains the best set of reagents and protocols to duplicate our results.

For cloning virus mRNA species, a host cell line sensitive to infection by the virus was infected at 5-10 plaque forming units per cell. When cytopathic effect was evident, but before total destruction, the medium was removed and the cells were lysed in 10 mls lysis buffer (4M guanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0, 0.5% N-lauroyl sarcosine, 0.1M beta-mercaptoethanol). The cell lysate was poured into a sterilized Dounce homogenizer and homogenized on ice 8-10 times until the solution was homogenous. For RNA purification, 8 mls of cell lysate were gently layered over 3.5 mls of CsCl solution (5.7M CsCl, 25 mM sodium citrate pH 7.0) in a Beckman SW41 centrifuge tube. The samples were centrifuged for 18 hours at 2° C. at 36,000 rpm in a Beckman SW41 rotor. The tubes were put on ice and the supernatants from the tubes were carefully removed by aspiration to leave the RNA pellet undisturbed. The pellet was resuspended in 400 microliters glass distilled water, and 2.6 mls of guanidine solution (7.5M guanidine-HCl, 25 mM sodium citrate pH 7.0, 5 mM dithiothreitol) were added. Then 0.37 volumes of 1M acetic acid were added, followed by 0.75 volumes of cold ethanol and the sample was put at -20° C. for 18 hours to precipitate RNA. The precipitate was collected by centrifugation in a Sorvall centrifuge for 10 min at 4° C. at 10,000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, recentrifuged at 13,000 rpm, and the supernatant saved. RNA was reextracted from the pellet 2 more times as above with 0.5 ml distilled water, and the supernatants were pooled. A 0.1 volume of 2M potassium acetate solution was added to the sample followed by 2 volumes of cold ethanol and the sample was put at -20° C. for 18 hours. The precipitated RNA was collected by centrifugation in the SS34 rotor at 4° C. for 10 minutes at 10,000 rpm. The pellet was dissolved in 1 ml distilled water and the concentration taken by absorption at A260/280. The RNA was stored at -70° C.

mRNA containing polyadenylate tails (poly-A) was selected using oligo-dT cellulose (Pharmacia #27 5543-0). Three milligrams of total RNA was boiled and chilled and applied to a 100 mg oligo-dT cellulose column in binding buffer (0.1M Tris pH 7.5, 0.5M LiCl, 5 mM EDTA pH 8.0, 0.1% lithium dodecyl sulfate). The retained poly-A⁺ RNA was eluted from the column with elution buffer (5 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 0.1% sodium dodecyl sulfate). This mRNA was reapplied to an oligo-dT column in binding buffer and eluted again in elution buffer. The sample was precipitated with 200 mM sodium acetate and 2 volumes cold ethanol at -20° C. for 18 hours. The RNA was resuspended in 50 microliters distilled water.

Ten micrograms poly-A⁺ RNA was denatured in 20 mM methyl mercury hydroxide for 6 minutes at 22° C. Beta-mercaptoethanol was added to 75 mM and the sample was incubated for 5 min at 22° C. The reaction mixture for first strand cDNA synthesis in 0.25 ml contained 1 microgram oligo-dT primer (P-L Biochemicals) or 1 microgram synthetic primer, 28 units placental ribonuclease inhibitor (Bethesda Research Labs #5518SA), 100 mM Tris pH 8.3, 140 mM KCl, 10 mM MgCl₂, 0.8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia), 100 microcuries ³² P-labelled dCTP (New England Nuclear #NEG-013H), and 180 units AMV reverse transcriptase (Molecular Genetics Resources #MG 101). The reaction was incubated at 42° C. for 90 minutes, and then was terminated with 20 mM EDTA pH 8.0. The sample was extracted with an equal volume of phenol/chloroform (1:1) and precipitated with 2M ammonium acetate and 2 volumes of cold ethanol -20° C. for 3 hours. After precipitation and centrifugation, the pellet was dissolved in 100 microliters distilled water. The sample was loaded onto a 15 ml G-100 Sephadex column (Pharmacia) in buffer (100 mM Tris pH 7.5, 1 mM EDTA pH 8.90, 100 mM NaCl). The leading edge of the eluted DNA fractions were pooled, and DNA was concentrated by lyophilization until the volume was about 100 microliters, then the DNA was precipitated with ammonium acetate plus ethanol as above.

The entire first strand sample was used for second strand reaction which followed the Gubler and Hoffman (23) method except that 50 micrograms/ml dNTP's, 5.4 units DNA polymerase I (Boerhinger Mannheim #642-711), and 100 units/ml E. coli DNA ligase (New England Biolabs #205) in a total volume of 50 microliters were used. After second strand synthesis, the cDNA was phenol/chloroform extracted and precipitated. The DNA was resuspended in 10 microliters distilled water, treated with 1 microgram RNase A for 10 minutes at 22° C., and electrophoresed through a 1% agarose gel (Sigma Type II agarose) in 40 mM Tris-acetate buffer pH 6.85. The gel was stained with ethidium bromide, and DNA in the expected size range was excised from the gel and electroeluted in 8 mM Tris-acetate pH 6.85. Electroeluted DNA was lyophilized to about 100 microliters, and precipitated with ammonium acetate and ethanol as above. The DNA was resuspended in 20 microliters water.

Oligo-dC tails were added to the DNA to facilitate cloning. The reaction contained the DNA, 100 mM potassium cacodylate pH 7.2, 0.2 mM dithiothreitol, 2 mM CaCl₂, 80 micromoles dCTP, and 25 units terminal deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 microliters. After 30 minutes at 37° C., the reaction was terminated with 10 mM EDTA, and the sample was phenol/chloroform extracted and precipitated as above.

The dC-tailed DNA sample was annealed to 200 ng plasmid vector pBR322 that contained oligo-dG tails (Bethesda Research Labs #5355 SA/SB) in 200 microliters of 0.01M Tris pH 7.5, 0.1M NaCl, 1 mM EDTA pH 8.0 at 65° C. for 2 minutes and then 57° C. for 2 hours. Fresh competent E. coli DH-1 cells were prepared and transformed as described by Hanahan (41) using half the annealed cDNA sample in twenty 200 microliter aliquots of cells. Transformed cells were plated on L-broth agar plates plus 10 micrograms/ml tetracycline. Colonies were screened for the presence of inserts into the ampicillin gene using Ampscreen (Bethesda Research Labs #5537 UA), and the positive colonies were picked for analysis.

POLYMERASE FILL-IN REACTION. DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KCl, 5 mM MgCl₂, and 400 micromolar each of the four deoxynucleotides. Ten units of Klenow DNA polymerase (BRL) were added and the reaction was allowed to proceed for 15 minutes at room temperature. The DNA was then phenol extracted and ethanol precipitated as above.

EXAMPLES Example 1

S-IBR-002

S-IBR-002 is an IBR virus that has a deletion of approximately 800 bp in the repeat region of the genome. This deletion removes the only two EcoRV restriction sites on the virus genome and an adjacent BglII site (FIG. 2).

To construct this virus, the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS was performed. Purified IBR virus DNA (Cooper strain) digested with EcoRV restriction enzyme was mixed with DraI-restriction enzyme-digested plasmid DNA containing the E.coli β-galactosidase (lacZ) gene under the control of the HSV-1 TK promoter. After ligation the mixture was used to transfect animal cells and the transfection stock was screened for recombinant IBR virus by the SOUTHERN BLOTTING OF DNA procedure. The final result of the purification was the recombinant IBR virus designated S-IBR-002. It was shown by Southern hybridization that this virus does not carry any foreign genes. Restriction enzyme analysis also showed that the insertion sites (EcoRV) in both repeats were deleted. FIG. 2 shows the restriction map of the EcoRI B fragment which contains the EcoRV restriction sites and the map of S-IBR-002 which lacks the EcoRV sites. S-IBR-002 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2140.

A study was conducted to determine the safety and serological response of young calves following intramuscular administration of S-IBR-002. These results are presented in Table 1. Three calves were inoculated intramuscularly with 10⁷ PFU of S-IBR-002. Clinical signs of IBR and febrile response were absent in these calves, as well as in the contact control calf. All three calves developed significant neutralizing antibody to IBR virus but the contact control remained seronegative. These results suggest that S-IBR-002 is useful as a vaccine against IBR disease.

                  TABLE 1                                                          ______________________________________                                         Serologic and Clinical Response of                                             Young Calves Following Vaccination with S-IBR-002                                                               Antibody Titer                                Virus  Calf   Clinical and                                                                               Virus  Days Post Inoculation                         Construct                                                                             #      Febrile response                                                                           Isolation.sup.a                                                                       0   7   14  21  28                            ______________________________________                                         S-IBR-002                                                                             28     NONE        (-)    <2  <4    6   5   3                                  30     NONE        (-)    <2  <4    6 <2    6                                  94     NONE        (-)    <2  <4    6   3   8                           Control                                                                               32     NONE        (-)    <4  <4  <4  <2  <4                            ______________________________________                                          .sup.a From nasal swabs and peripheral blood leukocytes.                 

Example 2

Unique Short 2 gene

The unique short region of IBR virus contains a gene homologous to the US2 gene of several other herpesviruses. In the studies described below we show that deletion of the IBR unique short 2 gene (US2) may render the virus safe for use in pregnant cows, as determined by direct fetal inoculation.

Observing that the Nasalgen IBR vaccine strain will not cause abortion when used in IBR-susceptible pregnant cows at various stages of gestation (18,65), we attempted to determine the genomic lesion responsible for this property. We characterized the genome of this virus by restriction mapping and DNA sequence analysis. It was determined that a major portion of the IBR virus US2 gene was deleted from the Nasalgen virus. Restriction mapping of the Nasalgen virus indicated that the HindIII K fragment contained an approximately 800 base pair deletion. The deletion was localized to the end of the HindIII K fragment located next to the HindIII O fragment (see FIG. 1). Therefore, the HindIII K fragment from the Cooper strain was subcloned and this region was sequenced. The first 1080 base pairs of the fragment were found to contain an open reading frame (ORF) coding for 309 amino acids (see FIG. 3). The ORF is 68% G+C and encodes a protein with a predicted molecular weight of 46,094. Comparison of the sequence of the predicted protein with sequences of gene products of HSV-1, PRV, HSV-2, and MDV in the unique short region indicated that this ORF is homologous to the herpesvirus US2 gene (see FIG. 4). Although the function of the herpesvirus US2 gene is not known, the gene has been shown to be nonessential for growth of HSV in cell culture (4,19). The US2 gene has also been shown to be deleted in the PRV vaccine strains Norden and Bartha (11).

The HindIII K fragment from the Nasalgen virus was subcloned and the deletion region was sequenced. When the sequence obtained from the Nasalgen strain was compared to the sequence obtained from the Cooper strain (see FIG. 5), it was possible to determine that amino acids 59 to 309 of the US2 gene had been deleted. It was also determined that most of the HindIII O fragment had also been deleted.

Cattle studies have shown that the Nasalgen virus will not cause abortion when used in IBR-susceptible pregnant cows at various stages of gestation (18). Since the only major difference between the wild-type IBR strain and the Nasalgen strain resides in the deletion of the US2 gene, this gene may be involved in the fetal virulence observed for the wild type virus.

Example 3

S-IBR-027

S-IBR-027 is an IBR virus that has a deletion of approximately 800 bp in the repeat regions and approximately 1200 bp in the short unique region of the genome. The deletion in the short unique region removes the US2 gene (FIG. 6). The repeat deletion was derived from the parental virus S-IBR-002 and is described in Example 2.

To construct this virus, the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS was performed. A homology vector containing the bacterial transposon Tn5 NEO (aminoglycosidase 3'-phosphotransferase) gene under the control of the HSV α4 promoter flanked by sequences from the IBR virus TK gene was constructed. The IBR virus homology regions were derived from the TK gene. The upstream homology included the first amino acid of the TK gene (15) and extended approximately 800 base pairs upstream of the TK coding region. The downstream homology included amino acids 156 to 357 and extended downstream of the TK coding region approximately 60 base pairs. S-IBR-002 DNA was mixed with the homology vector and transfected into rabbit skin cells as indicated in the methods. The transfection stock was selected according to the SELECTION OF G418 RESISTANT IBR VIRUS. Individual clones were picked after one round of selection and analyzed by the SOUTHERN BLOTTING OF DNA procedure. When a probe derived from the NEO gene was used in this analysis, one clone was found which did not hybridize to the NEO probe but had a HindIII restriction digestion pattern clearly distinct from the parental S-IBR-002. Further analysis indicated that the NEO had not been inserted into the TK region, however an approximately 1200 base pair deletion had occurred in the HindIII K fragment.

In order to characterize the HindIII K deletion, that fragment was subcloned and subjected to restriction mapping. Utilizing a series of oligonucleotide probes derived from the wild type sequence it was determined that approximately 1200 base pairs were deleted from the end of the HindIII K fragment adjacent to the HindIII K/HindIII O junction (see FIG. 6). This deletion removes the entire coding region of the US2 gene. S-IBR-027 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2322.

Direct fetal inoculation is the most sensitive test for determining the safety of live, IBR vaccines as regards their use in pregnant cows or in calves nursing pregnant cows. Three virus constructs were tested for fetal safety by inoculating directly into the bovine fetus, following laparotomy to expose the uterus. Abortion occurring within seven days after inoculation was considered to be surgically-induced. If fetuses aborted after this time, tissue samples were removed and cultured for the presence of the IBR construct. Caesarean sections were performed on cows with fetuses surviving for greater than 30 days post-inoculation. Fetal tissue was removed for virus culturing and blood samples were taken for evaluation of serum antibody to IBR virus.

The S-IBR-027 construct described above was tested, as well as two other constructs, S-IBR-020 and S-IBR-028. The S-IBR-020 construct was derived from the Cooper strain of IBR virus by making deletions in the repeat regions of the DNA and by inserting the Tn5 NEO gene. The S-IBR-028 construct was derived from the Cooper strain of IBR virus by making deletions in the repeat region of the DNA and in the TK gene. The Tn5 NEO gene was also inserted into the TK deletion.

The following results were obtained from studies with the three virus constructs. In the studies with S-IBR-020, two fetuses were inoculated, one at approximately 130-140 days gestation and the other at approximately 170-180 days gestation. The younger fetus aborted twenty days after inoculation, but virus could not be recovered from tissue samples of this fetus (Table 2). The other fetus was live and appeared normal when it was surgically removed 60 days post-inoculation. In studies with S-IBR-027, four fetuses, ranging in age from 125 days to >250 days, were inoculated (Table 2). All fetuses survived and appeared normal. In studies with S-IBR-028, three fetuses, ranging in age from 140 days to >250 days, were inoculated. The youngest and eldest fetuses survived and appeared normal, however the fetus inoculated at 160-170 days gestation aborted nine days after inoculation.

Direct fetal inoculation is the most sensitive test for measuring the safety of live, IBR viruses used in pregnant cows. To date, the gene(s) involved in fetal virulence has not been reported. We have engineered IBR viruses with deletions in three different regions of IBR virus DNA and then determined the effect of the gene deletion. All three virus constructs tested have a deletion in the repeat region of the DNA and two constructs do not have TK activity. One fetus inoculated with each of the TK- constructs has aborted. In contrast, the construct with deletions in the repeat regions and the US2 gene (S-IBR-027) has been inoculated into four fetuses with no adverse reactions.

                  TABLE 2                                                          ______________________________________                                         Safety of IBR Viruses for Bovine Fetuses                                       Construct  Fetal Age.sup.a                                                                             Results                                                ______________________________________                                         S-IBR-020  130-140 Days Fetus aborted Day 20                                                           post-inoculation; no                                                           virus isolated                                                    170-180 Days Normal, live fetus 60                                                          days post-inoculation                                  S-IBR-027  125-135 Days Normal, live fetus 60                                                          days post-inoculation                                             150-160 Days Normal, live calf born                                                         56 days post-inoculation                                          220-240 Days Normal, live calf born                                                         30 days post-inoculation                                          >250 Days    Normal, live calf born                                                         30 days post-inoculation                               S-IBR-028  140-150 Days Normal, live fetus 60                                                          days post-inoculation                                             160-170 Days Fetus aborted Day 9                                                            post-inoculation; no                                                           virus isolated                                                    >250 Days    Normal, live calf born                                                         12 days post-inoculation                               ______________________________________                                          .sup.a Approximate age at time of virus inoculation                      

We have shown that S-IBR-027 is safe for fetal inoculation in contrast to S-IBR-020 and S-IBR-028 which are not. Although all three viruses were engineered by similar approaches, the distinguishing difference of S-IBR-027 is the deletion of the US2 gene. We have also shown that the Nasalgen virus, which was generated by independent methods and is safe for use in IBR-susceptible pregnant cows, has been deleted in the US2 gene.

Although the S-IBR-027 and Nasalgen have the similar property of fetal safety, S-IBR-027 offers additional advantages. The major portion of the US2 gene (251 out of 309 amino acids) has been deleted in the Nasalgen virus. This deletion would clearly inactivate the gene, however the remaining portion of the gene may make it more likely to revert to virulence via recombination with other viruses. The complete coding region of the US2 has been deleted from S-IBR-027 making it less likely that this gene could be restored and revert the virus to virulence. The S-IBR-027 construct also carries an important deletion in the repeat region, which is not present in the Nasalgen virus. A deletion in the analogous region of the pseudorabies virus (PRV) has been shown to be valuable in attenuating PRV for swine (see U.S. Pat. No. 4,877,737). This deletion has also been shown to attenuate IBR for cattle as seen in the testing of S-IBR-002 (see Example 1).

Example 4

S-IBR-028

S-IBR-028 is an IBR virus that has a deletion of approximately 800 bp in the repeat regions and approximately 250 bp in the TK region of the genome. The deletion in the TK region inactivates the TK gene. The repeat deletion was derived from the parental virus S-IBR-002 and is described in Example 2.

To construct this virus, the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS was performed. A homology vector containing the bacterial transposon Tn5 NEO (aminoglycosidase 3'-phosphotransferase) gene under the control of the HSV-1 α4 gene promoter flanked by sequences from the IBR virus TK gene was constructed. The IBR virus homology regions were derived from the TK gene. The upstream homology included amino acids 1 to 62 of the TK gene (15) and extended approximately 674 base pairs upstream of the TK coding reigon. The downstream homology included amino acids 156 to 357 and extended downstream of the TK coding region approximately 1138 base pairs. S-IBR-002 DNA was mixed with the homology vector 129-71.5 and transfected into rabbit skin cells as indicated in the methods. The transfection stock was selected according to the SELECTION OF G418 RESISTANT IBR VIRUS.

Individual clones were picked after two rounds of selection and analyzed by the SOUTHERN BLOTTING OF DNA procedure. Several clones were assayed for TK activity by a ¹⁴ C-thymidine incorporation assay (29). One clone which was negative for TK activity was chosen and characterized by digestion with HindIII and XbaI. The restriction endonuclease analysis confirmed that the NEO gene had been inserted into the TK gene. This clone, designated S-IBR-028, has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2326.

Example 5

Glycoprotein G gene

Deletion of the PRV gpX gene has been shown to be valuable both as an attenuating lesion and as a negative serological marker (see U.S. Ser. No. 07/192,866, filed May 11, 1988). In the studies described below we show that the unique short region of IBR virus contains a gene homologous to the gpX gene of PRV.

The sequence of an approximately 1400 base pair region of the IBR HindIII K fragment (see FIG. 8), located approximately 2800 base pairs downstream of the HindIII K/HindIII O junction was determined. This region was found to contain an ORF coding for 441 amino acids translated in the direction away from the HindIII K/HindIII O junction (see FIG. 1). The ORF is 69% G+C and encodes a protein with a predicted molecular weight of 58,683. Comparison of the sequence of the predicted protein with sequences of gene products of HSV-2 and PRV in the unique short region indicated that this ORF is homologous to the herpesvirus gpG gene (see FIG. 9). The complete gpG gene resides on an approximately 2800 base pair MluI to NdeI sub-fragment of the IBR virus HindIII K fragment. This sub-fragment has been cloned as a blunt ended fragment into the plasmid pSP64. This plasmid is designated PSY1643. PSY1643 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. 68652. This plasmid may be used to confirm the sequence of the gpG gene. The sequence of the gpG gene may also be confirmed by comparing the appropriate DNA sequence of the wild type virus S-IBR-000 (Cooper Strain) with the sequence of the gpG deleted virus S-IBR-037 (ATCC Accession No. 2320).

To confirm the expression of the IBR virus gpG gene product, cells were infected with IBR virus and samples of media from infected cultures were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. The anti-serum used was a mouse hyper-immune serum raised against chemically-synthesized gpG peptides (amino acids 242-254 and 269-289) linked to keyhole limpet hemocyanin. As shown in FIG. 10, gpG is prominent in the media of cells infected with wild type virus (S-IBR-000), but is not detected in media of mock infected cells.

Example 6

S-PRV-160

S-PRV-160 is a pseudorabies virus that has a deletion in the TK gene in the long unique region, a deletion in the repeat region, and an approximately 1414 base pair deletion in the gpX coding region. The gene for E.coli β-galactosidase (lacZ gene) was inserted in the place of the gpX gene and is under the control of the gpX promoter. A chimeric gene coding for an IBR virus gpG, PRV gpIII and PRV gpX fusion protein was inserted at the HindIII sites located in each repeat.

S-PRV-160 was constructed utilizing plasmid 459-12.6, pseudorabies virus S-PRV-013 (see U.S. Ser. No. 823,102, filed Jan. 27, 1986 and U.S. Ser. No. 07/192,866, filed May 11, 1988) and the restriction enzyme HindIII in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Several clones were screened by digestion with HindIII for the presence of the HindIII band containing the chimeric gene insert from plasmid 459-12.6. One clone exhibiting the correct HindIII insert band was chosen and designated S-PRV-160.

S-PRV-160 was constructed so that it would express precisely the gpG specific amino acids that were deleted in S-IBR-037. This allows the gpG fusion protein expressed in S-PRV-160 to be used as an antigen to identify antibodies directed against the wild type virus as opposed to antibodies directed against S-IBR-037. Note that gpX, the PRV homologue of IBR virus gpG, has been deleted from S-PRV-160 to prevent any confusion resulting from cross reactivity that might exist between the two proteins. To confirm that S-PRV-160 does express IBR virus gpG, a Western blot analysis was performed. As can be seen in FIG. 10, gpG specific antibody does react with an appropriately sized media protein from S-PRV-160.

S-PRV-160 may also be utilized as an antigen for the production of gpG specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the gpG protein. Monoclonal antibodies were generated in mice utilizing S-PRV-160 according to the PROCEDURE FOR GENERATING MONOCLONAL ANTIBODIES. One of these antibodies, clone 3-1G7, was shown to react specifically with purified gpG in the gpG ELISA assay.

Example 7

S-IBR-035

S-IBR-035 is an IBR virus that has two deletions in the short unique region of the genome. The first deletion is approximately 2500 base pairs and begins in the HindIII K fragment approximately 1750 base pairs downstream of the HindIII O/HindIII K junction and extends back through that junction. This deletion removes the US2 gene. The second deletion is approximately 294 base pairs and begins in the HindIII K fragment approximately 3900 base pairs downstream of the HindIII K/HindIII O junction and extends back toward that junction. This deletion removes amino acids 263 to 361 of the gpG gene. The gene for E.coli β-galactosidase (lacZ gene) was inserted into the deletion in the gpG gene and is under the control of the HCMV immediate early promoter.

S-IBR-035 was derived from S-IBR-000 (Cooper strain). This was accomplished utilizing the homology vector 439-01.31 (see Materials and Methods) and virus S-IBR-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. The final result of blue plaque purification was the recombinant virus designated S-IBR-035. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the β-galactosidase (lacZ) marker gene and the deletion of approximately 294 base pairs of the gpG gene. It was also confirmed that an approximately 2500 base pair deletion had occurred in the region of the US2 gene.

Example 8

S-IBR-036

S-IBR-036 is an IBR virus that has two deletions in the short unique region of the genome. The first deletion is approximately 2500 base pairs and is similar to the deletion in S-IBR-035 (see Example 7) which removes the US2 gene. The second deletion is approximately 1230 base pairs and begins in the HindIII K fragment approximately 3900 base pairs downstream of the HindIII O/HindIII K junction and extends back toward that junction. This deletion removes amino acids 1 to 361 of the gpG gene. The gene for E.coli β-galactosidase (lacZ gene) was inserted into the deletion in the gpG gene and is under the control of the HCMV immediate early promoter.

S-IBR-036 was derived from S-IBR-000 (Cooper strain). This was accomplished utilizing the homology vector 439-21.69 (see Materials and Methods) and virus S-IBR-000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. The final result of blue plaque purification was the recombinant virus designated S-IBR-036. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the β-galactosidase (lacZ) marker gene and the deletion of approximately 1230 base pairs of the gpG gene. It was also confirmed that an approximately 2500 base pair deletion had occurred in the region of the US2 gene (see above).

Example 9

S-IBR-037

S-IBR-037 is an IBR virus that has two deletions in the short unique region of the genome. The first deletion is approximately 2500 base pairs and begins in the HindIII K fragment approximately 1750 base pairs downstream of the HindIII O/HindIII K junction and extends back through that junction. This deletion removes the US2 gene. The second deletion is approximately 1230 base pairs and begins in the HindIII K fragment approximately 3900 base pairs downstream of the HindIII O/HindIII K junction and extends back toward that junction. This deletion removes amino acids 1 to 361 of the gpG gene.

S-IBR-037 was derived from S-IBR-035. This was accomplished utilizing the homology vector 439-70.4 (see Materials and Methods) and virus S-IBR-035 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS. The result of white plaque purification was the recombinant virus designated S-IBR-037. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the deletion of the β-galactosidase (lacZ) marker gene and the deletion of approximately 1230 base pairs of the gpG gene. It was also confirmed that an approximately 2500 base pair deletion had occurred in the region of the US2 gene (see above). S-IBR-037 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2320. To test the efficacy of S-IBR-037 as an inactivated IBR virus vaccine in protecting susceptible calves against virulent IBR virus challenge, a study was performed according to the VACCINATION STUDIES IN CALVES WITH INACTIVATED IBR VIRUS. The following results were observed.

Virus neutralization antibody titers were elicited in animals after the first vaccination (see Table 3). Antibody titers were not significantly different between animals that received a vaccine dose of 10⁷.3 virus and animals vaccinated with 10⁸.0 virus. After the second vaccination, mean antibody titers increased to 1:19 and 1:32, respectively, for the 10⁷.3 and 10⁸.0 vaccine groups. Control animals remained seronegative to IBR virus throughout the vaccination period. Antibody titers in both vaccinate groups showed an increase typical of an anamnestic response after challenge with virulent IBR virus. By 13 days post challenge, mean antibody titers were 1:152 and 1:215 for the 10⁷.3 and 10⁸.0 vaccinate groups respectively. In contrast, mean antibody titers in challenged control animals were 1:4 at 7 days and 1:8 at 13 days post challenge.

Nasal swabs were collected from challenged animals to determine whether vaccination decreased the time of virus shedding (Table 4). The most dramatic difference between vaccinates and control animals was observed at 12 days post challenge. At this time, seventy-five percent of controls animals continue to shed, whereas, only twenty-five percent of both vaccinate groups shed virus. Virus was not isolated from control or vaccinated groups at 15 days post challenge.

                  TABLE 3                                                          ______________________________________                                         Generation of virus neutralizing antibody in animals                           vaccinated with inactivated S-IBR-037 vaccine.                                 Antibody titer.sup.a on days:                                                  Post Vaccination             Post Challenge                                    Animal No.                                                                             7      21     28     42      7     13                                  ______________________________________                                         Controls                                                                        9      ≦2                                                                             ≦2                                                                             ≦2                                                                             ≦2                                                                              4     4                                   22      ≦2                                                                             ≦2                                                                             ≦2                                                                             ≦2                                                                              4     8                                   32      ≦2                                                                             ≦2                                                                             ≦2                                                                             ≦2                                                                              4     16                                  64      ≦2                                                                             ≦2                                                                             ≦2                                                                             ≦2                                                                              4     8                                   GMT     ≦2                                                                             ≦2                                                                             ≦2                                                                             ≦2                                                                              4     8                                   Vaccinates                                                                     dose 10.sup.7.3                                                                 1      ≦2                                                                             8      32     64      64    128                                 20      ≦2                                                                             8      32     64      64    256                                 25      ≦2                                                                             8      16      8      64    512                                 36      ≦2                                                                             4      16      4      16    ≧32                          GMT     ≦2                                                                             6.7      22.6*                                                                                 19.0* 45.34*                                                                               152.2*                              Vaccinates                                                                     dose 10.sup.8.0                                                                 7      ≦2                                                                             4      32      8      64    256                                 30      ≦2                                                                             ≧8                                                                             64     128     128   ≧128                         33      ≦2                                                                             16     32     128     128   256                                 69      ≦2                                                                             4      16      8      128   256                                 GMT     ≦2                                                                             6.7     32*    32*    107.6*                                                                               215.3*                              ______________________________________                                          *Statistically greater than controls (p ≦ 0.05)                         .sup.a Expressed as reciprocal of dilution.                              

                  TABLE 4                                                          ______________________________________                                         Isolation of IBR virus from vaccinated and unvaccinated control                animals after challenge with virulent IBR virus.                                       IBR virus isolated (+/-) from animals                                          on days post challenge                                                 Animal No.                                                                               3         6      9      12   15                                      ______________________________________                                         Controls                                                                        9        -         +      +      +    -                                       22        -         +      +      -    -                                       32        -         +      +      +    -                                       64        -         +      +      +    -                                       Vaccinates                                                                     dose 10.sup.7.3                                                                 1        -         +      +      -    -                                       20        -         +      +      -    -                                       25        -         +      +      -    -                                       36        -         +      +      +    -                                       Vaccinates                                                                     dose 10.sup.8.0                                                                 7        -         +      +      -    -                                       30        -         -      -      -    -                                       33        -         +      +      +    -                                       69        -         +      +      -    -                                       ______________________________________                                    

                  TABLE 5                                                          ______________________________________                                         Vaccinated animals demonstrate reduced clinical signs of IBR.                  Clinical scores post challenge                                                                                 Muco-                                                           Nasal   Serous purulent                                                                               Tempera-                               Animal No.                                                                             Attitude.sup.a                                                                          Ulcers.sup.b                                                                           Discharge.sup.c                                                                       Discharge.sup.d                                                                        ture.sup.e                             ______________________________________                                         Controls                                                                        9      5        3       11     5       3                                      22      2        2       12     3       1                                      32      5        3       11     0       4                                      64      6        3       11     1       1                                      GMS     4.5      2.8     11.3   2.3     2.3                                    Vaccinates                                                                     dose 10.sup.7.3                                                                 1      0        2       1      0       0                                      20      0        1       3      0       0                                      25      0        2       6      2       0                                       36*    6        2       1      13      0                                      GMS     1.5      1.8     2.8*   2.3     0                                      Vaccinates                                                                     dose 10.sup.8.0                                                                 7      1        2       1      0       0                                      30      1        2       2      2       0                                      33      1        2       0      0       0                                      69      1        2       0      0       0                                      GMS     1        2       0.8*   0.5     0                                      ______________________________________                                          .sup.a Days with depressed attitude.                                           .sup.b Number of ulcers.                                                       .sup.c Days with serous discharge.                                             .sup.d Days with mucopurulent discharge.                                       .sup.e Days with ≧2° F. above baseline temperature.              .sup.f Animal exhibited mucopurulent discharge on the day of challenge an      for 13 days post challenge.                                                    *Statistically different from controls (p ≦ 0.05)                 

Animals were observed daily for 13 days post challenge for clinical signs of IBR infection. Clinical disease was evaluated with respect to attitude, the number of ulcers, extent of serous and mucopurulent discharge and the number of days with elevated temperature. The results presented in Table 5 show that vaccinated animals exhibited less severe disease than did unvaccinated control animals. Control animals showed clinical depression ("Attitude" in Table 5) for 4.5 days compared with 1 to 1.5 days for vaccinated animals. The amount and extent of serous discharge was substantially reduced in both vaccinate groups compared with controls. The extent of mucopurulent discharge was also reduced in vaccinated animals, although to a lesser degree. However, vaccinate animal #36 did have mucopurulent discharge on the day of challenge and is not consistent with the results for other vaccinates. None of the vaccinates exhibited temperatures of ≧2° F. above baseline. In contrast, all control animals exhibited elevated temperatures of ≧2° F. over baseline and 2 of 4 control animals had temperatures of 104° F. and above.

Vaccination of calves with inactivated S-IBR-037 vaccine protected the animals against virulent wild-type IBR virus challenge. Virus neutralization titers were statistically greater in vaccinated than in control animals. An anamnestic response in antibody titer was observed 7 days post challenge, indicating the development of humoral memory response. Except for 7 days post challenge, neutralization titers between the 10⁷.3 and 10⁸.0 vaccinate groups were not statistically different. Fewer vaccinated animals shed virulent challenge virus than control animals. These results suggest that virulent IBR virus is cleared more rapidly in vaccinated than in unvaccinated animals. Clinical symptoms of IBR virus infection were also reduced in vaccinated animals. After challenge, both vaccinate groups exhibited fewer days of depressed attitude, reduced serous discharge, and no elevated temperature compared with controls.

In order to show that gpG antibody is produced in vaccinated calves following exposure to wild-type virus, serum samples taken pre- and post-exposure to wild-type virus were subjected to the ELISA assay. Samples taken at the day of challenge and at 13 days post-challenge were analyzed. As seen in Table 6, the post-challenge absorbance readings for gpG increase for each animal (ratio of >1.0), indicating that within 13 days of infection a detectable immune response to gpG is present.

                  TABLE 6                                                          ______________________________________                                         Detection of antibody to gpG in serum of animals                               vaccinated with S-IBR-037 and challenged with wild type.                       Animal No.   Ratio of pre- vs. post challenge.sup.a                            ______________________________________                                         Controls                                                                        9           1.22                                                              22           1.96                                                              32           1.87                                                              64           2.19                                                              Vaccinates dose                                                                10.sup.7.3                                                                      1           1.39                                                              20           1.40                                                              25           1.84                                                              36           1.18                                                              Vaccinates dose                                                                10.sup.8.0                                                                      7           1.19                                                              30           1.29                                                              33           1.52                                                              69           2.66                                                              ______________________________________                                          .sup.a Animals were challenged with 10.sup.7.6 PFU of wild type IBR virus      Prechallenge serum from day of challenge, postchallenge serum from 13 day      post challenge. Data reflects the average of the ratio of absorbance           readings for three independent ELISA determinations.                     

Example 10

S-IBR-038

S-IBR-038 is an IBR virus that has two deletions in the short unique region of the genome. The first deletion is approximately 2500 base pairs and begins in the HindIII K fragment approximately 1750 base pairs downstream of the HindIII O/HindIII K junction and extends back through that junction. This deletion removes the US2 gene. The second deletion is approximately 294 base pairs and begins in the HindIII K fragment approximately 3900 base pairs downstream of the HindIII K/HindIII O junction and extends back toward that junction. This deletion removes amino acids 263 to 361 of the gpG gene.

S-IBR-038 resulted from the removal of the marker gene from S-IBR-035 (see above). This was accomplished by digestion of S-IBR-035 with XbaI as described in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The structure of S-IBR-035 was confirmed by restriction enzyme analysis with HindIII, BamHI and XbaI.

Example 11

Glycoprotein E gene

Deletion of the PRV gI gene has been shown to be valuable both as an attenuating lesion and a negative serological marker (3,42). In the studies described below we show that the unique short region of IBR virus contains a gene homologous to the gI gene of PRV.

The sequence of 2038 base pairs of the IBR unique short region, starting approximately 1325 base pairs upstream of the HindIII K/HindIII F junction in the HindIII K fragment was determined. This region was found to contain an ORF coding for 617 amino acids translated in the direction away from the HindIII K/HindIII O junction (see FIG. 1). The ORF is 70.5% G+C and encodes a protein with a predicted molecular weight of approximately 88,980. Comparison of the sequence of the predicted protein with sequences of gene products of HSV-1, VZV, and PRV in the unique short region indicated that this ORF is homologous to the herpesvirus gpE gene (see FIG. 16).

The DNA encoding the gpE gene has been cloned in two plasmids, PSY1644 and PSY1645. The amino-terminal half of the gene (encoding amino acids 1-276) was cloned as an approximately 2300 base pair fragment resulting from a partial SmaI digest of wild type S-IBR-000 (Cooper Strain) DNA. This fragment was inserted into the plasmid pSP64 to yield PSY1644. This plasmid, designated PSY1644, has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. 68651. The carboxyl-terminal half of the gene (encoding amino acids 277-617) was cloned as an approximately 2400 base pair SmaI fragment. This fragment was inserted into the plasmid pSP64 to yield PSY1645. This plasmid, designated PSY1645, has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. 68650. These plasmids may be used to confirm the sequence of the gpE gene.

Example 12

Pseudorabies virus expressing IBR virus gpE

A pseudorabies virus analogous to S-PRV-160 may be constructed for the purpose of expressing the IBR virus gpE. This may be accomplished by inserting the gene coding for IBR virus gpE into S-PRV-002 (U.S. Pat. No. 4,877,737).

Such an expression vector may be constructed utilizing the IBR virus gpE plasmid described in the methods section, pseudorabies virus S-PRV-002 and the restriction enzyme XbaI in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. Viruses resulting from this procedure may be screened by digestion with XbaI for the presence of the XbaI band containing the IBR virus gpE gene.

The gpE protein expressed from this vector may be used as an antigen to identify antibodies directed against the wild type virus as opposed to antibodies directed against gpE deleted viruses. This virus may also be utilized as an antigen for the production of gpE specific monoclonal antibodies. Such antibodies are useful in the development of diagnostic tests specific for the gpE protein. Monoclonal antibodies may be generated in mice utilizing this virus according to the PROCEDURE FOR GENERATING MONOCLONAL ANTIBODIES.

Example 13

Glycoprotein E deleted IBR viruses

The HOMOLOGY VECTOR 536-03.5 was used to generate various gpE-deleted IBR viruses. Utilizing the general strategy described in CONSTRUCTION OF DELETION VIRUSES, a gpE deletion of approximately 1410 base pairs (amino acids 77-547) was introduced into two different IBR virus backbones, S-IBR-000 (Cooper Strain) and S-IBR-037. The virus resulting from the S-IBR-000 parent contains the gpE deletion alone. The virus resulting from the S-IBR-037 parent contains the gpE deletion in conjunction with the US2 and gpG deletions. The lacZ marker gene may be removed from these viruses utilizing the procedures outlined in the methods section.

These gpE-deleted viruses are of great value as IBR vaccines. Their combination of different deletions will provide the varying degrees of attenuation which are required for a superior vaccine. These viruses will also provide a negative serological marker which may be used to distinguish vaccinated from infected animals. The virus containing both gpG and gpE deletions should be of even greater value by having two negative markers. The availability of two negative markers permits one marker to be used as a confirmatory test, greatly increasing the reliability of such a diagnostic determination.

Example 14

S-IBR-004

S-IBR-004 is an IBR recombinant virus carrying an inserted foreign gene, Tn5 NEO (aminoglycoside 3'-phosphotransferase) gene, under the control of the pseudorabies virus (PRV) glycoprotein X promoter.

To construct this virus, the HindIII K DNA fragment from wild type IBR virus was cloned into the plasmid pSP64 at the HindIII site. This plasmid was designated pSY524. A map of the HindIII K fragment is shown in FIG. 19. The DNA from the XhoI site to the HindIII site and containing the NdeI site from pSY524 was cloned into plasmid pSP65 and called pSY846. The NdeI to EcoRI fragment was removed from pSY846 by digestion with NdeI and EcoRI restriction enzymes, followed by POLYMERASE FILL-IN REACTION and LIGATION. The resulting plasmid was called pSY862. The plasmid pNEO (P.L. Biochemicals, Inc.) contains the aminoglycoside 3'-phosphotransferase (NEO) gene and confers resistance to ampicillin and neomycin on E. coli hosts. The coding region of this gene (BglII-BamHI fragment) was isolated and cloned between the PRV gpX promoter and the HSV-TK poly A sequence in a plasmid called pSY845.

The NEO gene construct in pSY845 was excised with HindIII, made blunt ended by the POLYMERASE FILL-IN REACTION, and cloned into the SacI site of plasmid pSY862. The final product was called pSY868.

Wild type IBR DNA was mixed with pSY868 DNA and the mixture was transfected into rabbit skin cells to generate recombinant IBR. The recombinant IBR virus carrying a functional NEO gene was then isolated and purified according to the SELECTION OF G418 RESISTANT IBR VIRUS method.

S-IBR-004 recombinant IBR was shown to express the NEO gene by the fact that cells infected with this virus were resistant to the toxicity of G418. A detailed map of the plasmid construction is shown in FIG. 19. The structure of S-IBR-004 is also shown in FIG. 19. S-IBR-004 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2134.

Example 15

S-IBR-008

S-IBR-008 is an IBR virus that has a deletion in the short unique region, and an insertion of the bovine rotavirus glycoprotein 38 (gp38) gene in the XbaI site in the long unique region.

The bovine rotavirus gp38 gene was cloned utilizing the METHOD FOR cDNA CLONING BOVINE ROTAVIRUS gp38 GENE. The bovine rotavirus gp38 gene was then engineered to contain herpesvirus regulatory signals as shown in FIG. 20. This was accomplished by cloning the gp38 gene BamHI fragment contained in pSY1053 between the BamHI and BglII sites in pSY1052. The resulting plasmid, pSY1023, contained the PRV gpX promoter in front of the gp38 gene, and the HSV-1 TK polyadenylation signal behind the gp38 gene. The entire construct was flanked by XbaI sites to allow for the insertion of the XbaI fragment into IBR virus by direct ligation.

S-IBR-004 was the starting virus for the generation of S-IBR-008. S-IBR-004 DNA and pSY1023 DNA were mixed together, cut with XbaI, and transfected into rabbit skin cells according to the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened for recombinant virus by the ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS procedure using antibodies prepared against the rotavirus gp38 protein.

One of the viruses purified by this screen was S-IBR-008, which has the following characteristics. It contains the rotavirus gp38 gene plus the plasmid DNA inserted into the XbaI site in the long unique region of the virus genome, but no longer contains the NEO gene of parent S-IBR-004 in the unique short region. In fact, a small deletion was created in the unique short region at the location of the NEO gene, as evidenced by the absence of an XbaI site at this location in S-IBR-008.

S-IBR-008 was shown to be expressing the rotavirus gp38 gene by analysis of RNA transcription in infected cells, and by the ANTIBODY SCREEN FOR RECOMBINANT HERPESVIRUS procedure using antibodies specific for the gp38 gene. S-IBR-008 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2141. The structure of S-IBR-008 is shown in FIG. 20.

Example 16

S-IBR-018

S-IBR-018 is an IBR virus that has three foreign genes inserted: the E.coli beta-galactosidase gene and the neomycin resistance gene in the XbaI site in the unique long region, and the parainfluenza-3 (PI-3) virus hemaglutinin gene (HN) in the HindIII site in the unique long region immediately adjacent to the XbaI site.

For cloning the PI-3 HN gene, the SF-4 strain of PI-3 was grown in MADIN-DARBY bovine kidney (MDBK) cells in culture and RNA was extracted from infected cells. The RNA was used in a reverse transcription protocol as outlined in the cDNA CLONING procedure using poly-dT as primer for reverse transcriptase. From this procedure, a series of clones was obtained that comprised parts of the genome of the PI-3 virus. The location of the gene for the human PI-3 HN gene has been published (25,26) and this information was used to locate the gene in applicants' bovine PI-3 clones. The entire open reading frame of the bovine PI-3 HN gene was sequenced by applicants and is given in FIG. 21.

The HSV alpha-4 promoter was used to express the PI-3 HN gene and the HSV TK poly-A signal was used to terminate transcription. The engineering of this construct was done as shown in FIG. 22 A and B. The construct contained (5' to 3') the HSV ICP4 promoter, the ICP4 TATA box, the ICP4 cap site, a fusion within the ICP4 5' untranslated region to the PI-3 HN gene at the HhaI site, the HN gene start codon, the HN structural gene, the HN stop codon, a fusion within the HN 3' untranslated region to the HSV TK untranslated 3' region, and the HSV TK poly-A signal sequence.

This plasmid also contained the beta-galactosidase (lacZ) gene under the control of the PRV gpX promoter with the gpX poly-A termination signal, as well as the neomycin resistance gene under the control of the gpX promoter with the TK poly-A termination signal. These latter two genes were cloned in tandem at the XbaI site in BamHI C fragment (FIG. 22 A and B). This BamHI C fragment contained the homology regions for use in the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure. After the transfection step in the procedure, the resulting recombinant virus from the transfection stock was selected for by the SELECTION OF G418 RESISTANT IBR VIRUS procedure, followed by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure, and subsequently analyzed for the insertion of the PI-3 HN gene by the SOUTHERN BLOTTING OF DNA procedure. The virus that resulted from this screening was designated S-IBR-018.

The structure of S-IBR-018 is shown in FIG. 22 C.

Example 17

S-IBR-019

S-IBR-019 is an IBR virus that has three foreign genes inserted: the E. coli beta-galactosidase (lacZ) gene and the neomycin resistance gene in the XbaI site in the unique long region, and the parainfluenza-3 (PI-3) virus fusion gene (F) in the HindIII site in the long unique region adjacent to the XbaI site.

For cloning the PI-3 F gene, the SF-4 strain of PI-3 was grown in MDBK cells in culture and RNA was extracted from infected cells. The RNA was used in a reverse transcription protocol as outlined in the cDNA CLONING procedure using poly-dT as primer for reverse transcriptase. From this procedure, a series of clones was obtained that comprised parts of the genome of the PI-3 virus. The location of the gene for the Sendai virus F gene has been published (27) and this comparative sequence information was used to locate the homologous gene in applicants' bovine PI-3 clones.

The HSV alpha-4 promoter was used to express the PI-3 F gene and the HSV TK poly-A signal was used to terminate transcription. The construct contained (5' to 3') the HSV alpha-4 promoter, the alpha-4 TATA box, the alpha-4 cap site, a fusion in the alpha-4 5' untranslated region to the PI-3 F gene, the F start codon, the F structural gene, the F stop codon, a fusion in the F 3' untranslated region to the HSV TK 3' untranslated region, and the TK poly-A signal sequence.

This plasmid also contained the beta-galactosidase (lacZ) gene under the control of the PRV gpX promoter with the gpX poly-A termination signal, as well as the neomycin resistance gene under the control of the gpX promoter with the TK poly-A termination signal. These latter two genes were cloned in tandem at the XbaI site in BamHI-C fragment (FIG. 23 A and B). This BamHI-C fragment contained the homology regions for use in the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS procedure. After the transfection step in the procedure, the resulting recombinant virus from the transfection stock was selected for by the SELECTION OF G418 RESISTANT HERPESVIRUS procedure, followed by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure, and subsequently analyzed for the insertion of the PI-3 F gene by SOUTHERN BLOTTING OF DNA procedure. The virus that resulted from this screening was designated S-IBR-019.

The structure of S-IBR-019 is shown in FIG. 23 C.

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    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 51                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1079 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bovine Herpesvirus-1 (IBR Virus)                                 (B) STRAIN: Cooper                                                             (C) INDIVIDUAL ISOLATE: S-IBR- 000                                             (viii) POSITION IN GENOME:                                                     (B) MAP POSITION:  86.8 to  87.8                                               (C) UNITS: %G                                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 25..951                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        TTAAGCGTTGCCGTGGCGGTCGCCATGGTGACTATAGTCACGTGTGGCCGG51                          MetValThrIleValThrCysGlyArg                                                    15                                                                             ATAGGCGCGGCGCCTTCCAGGCAAGCCCAGACGTGCGCCGCGCGGGTG99                             IleGlyAlaAlaProSerArgGlnAlaGlnThrCysAlaAlaArgVal                               10152025                                                                       TGGCGTTTCCTTGCCGAGCAGAGCCGGGCGCTGACGGCAAGCCGGCTG147                            TrpArgPheLeuAlaGluGlnSerArgAlaLeuThrAlaSerArgLeu                               303540                                                                         GGGACGACGGTCGTTGTCTTCGATCACGCCCTAGTAAAAACGGCGAAG195                            GlyThrThrValValValPheAspHisAlaLeuValLysThrAlaLys                               455055                                                                         GGCTGCACGTCGACGTCAACGTCAAGCCAGCGGCGCGGGTGGCTTTTG243                            GlyCysThrSerThrSerThrSerSerGlnArgArgGlyTrpLeuLeu                               606570                                                                         TCGACACAGCGCCCTTGGCCCGGGCGCCGGCTTAGCCCGCCACCGCCA291                            SerThrGlnArgProTrpProGlyArgArgLeuSerProProProPro                               758085                                                                         ACCGGCGAGTGGGTCAGCTGGTCGACGGCTACAAACTTGCTGAAACTC339                            ThrGlyGluTrpValSerTrpSerThrAlaThrAsnLeuLeuLysLeu                               9095100105                                                                     GGCCGCGCGAGGGCTCGGCCCTTCCACATGTGGGTTTTTGGCGCCGCC387                            GlyArgAlaArgAlaArgProPheHisMetTrpValPheGlyAlaAla                               110115120                                                                      GATTTGTACGCGCCTATTTTTGCGCACATTGCCGCCACGACGCGCTTG435                            AspLeuTyrAlaProIlePheAlaHisIleAlaAlaThrThrArgLeu                               125130135                                                                      GTTTACGCGCAGCTGGACTGTACGTTTGCGGGAGCGGCGTGGCGGCTC483                            ValTyrAlaGlnLeuAspCysThrPheAlaGlyAlaAlaTrpArgLeu                               140145150                                                                      CCGCGGCGCGGCCCGGCCATCGCTAGCCCGTGGCCGCCCTACGATACC531                            ProArgArgGlyProAlaIleAlaSerProTrpProProTyrAspThr                               155160165                                                                      CCGACACTCCCTGAGCTGGTGGCCGGTGGTGTCCTTTTCCGGCTGGTC579                            ProThrLeuProGluLeuValAlaGlyGlyValLeuPheArgLeuVal                               170175180185                                                                   TACGAAGTCGTAGACCGCGGGCGGCGCCCCGCCCCGCCAAACGCGAGC627                            TyrGluValValAspArgGlyArgArgProAlaProProAsnAlaSer                               190195200                                                                      CCCCGTGCCCCAGGGGCTCGCCCCCGCGCGCGCCATGTGCTATCCTTT675                            ProArgAlaProGlyAlaArgProArgAlaArgHisValLeuSerPhe                               205210215                                                                      AAAGGCCGCACCCAGCGCCGGCGTTTGGTCATTTGCTTTGTGACCGCG723                            LysGlyArgThrGlnArgArgArgLeuValIleCysPheValThrAla                               220225230                                                                      CCGAGGGACCATGTTCCGCCAGGGCACCCCCAACCGCGTGGTGATCAG771                            ProArgAspHisValProProGlyHisProGlnProArgGlyAspGln                               235240245                                                                      CACAGTGCCGTTGAGCAGAGAGGCGACCGCGACCGCGACCGCCGGCAC819                            HisSerAlaValGluGlnArgGlyAspArgAspArgAspArgArgHis                               250255260265                                                                   CGGTCCCGGATGCGAGGGGGGGCTTGGTGGCTGGCGACTCTTTACAGT867                            ArgSerArgMetArgGlyGlyAlaTrpTrpLeuAlaThrLeuTyrSer                               270275280                                                                      GCCGCCACGAGCAAGAAGACGGCCTGTATGCTATCGTCCCGCCGGACT915                            AlaAlaThrSerLysLysThrAlaCysMetLeuSerSerArgArgThr                               285290295                                                                      ATTTTCCGGTGGTGCCCTCGTCCAAGCCCCTGCTGGTGAAAGTTCC961                              IlePheArgTrpCysProArgProSerProCysTrp                                           300305                                                                         CGCTCCCGGCGCGAGTCCCGACCGAACTGGGGGCGCAGTTCACTTTGAATGTGTTCCCGC1021               GCCGCGCCGACCGCTGCAGTTCTTTCGTCAGCTTTACGACGGTTCATTCGTTAAGCTT1079                 (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        HisMetTrpValPheGlyAlaAlaAspLeuTyrAlaProIlePheAla                               151015                                                                         HisIle                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Herpes Simplex Virus Type 1                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        HisLeuTrpValValGlyAlaAlaAspLeuCysValProPheLeuGlu                               151015                                                                         TyrAla                                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pseudorabies Virus                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        HisLeuTrpIleLeuGlyAlaAlaAspLeuCysAspGlnValLeuLeu                               151015                                                                         AlaAla                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Herpes Simplex Virus Type 2                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        HisLeuTrpValValGlyAlaAlaAspLeuCysValProPhePheGlu                               151015                                                                         TyrAla                                                                         (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Marek's Disease Virus                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        HisSerLeuTrpIleValGlyAlaAlaAspIleCysArgIleAlaLeu                               151015                                                                         GluCysIle                                                                      (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 60 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        TGAGCGCGCGCCGCTGCATGCTGGTGCGAACTCACGCCGAGCGCGCGTGCGAGCAAGCTT60                 (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 60 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        CTAGTAAAAACGGCGAAGGGCTGGTGCGAACTCACGCCGAGCGCGCGTGCGAGCAAGCTT60                 (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 60 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        CTAGTAAAAACGGCGAAGGGCTGCACGTCGACGTCAACGTCAAGCCAGCGGCGCGGGTGG60                 (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 48 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       GATTTAGGTGACACTATAGAATACACGGAATTCGAGCTCGCCCCATGG48                             (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 99 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..12                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       TTAAGTGGGATCCCGGCGCGCAGGCGCGCACGTCGGTCGCGGTCGCGCGCCA52                         LeuSerGlyIle                                                                   TGGGGGATCCTCTAGAGCTTGGGCTGCAGGTCCTGATTGATACACTG99                              (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 99 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       GCCCCGATCGTCCACACGGAGCGCGGCTGCCGACACGGATCTGATCAAGAGACAGGATGA60                 GGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCA99                                      MetIleGluGlnAspGlyLeuHisAla                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 99 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 64..78                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       GGACCTTGCACAGATAGCGTGGTCCGGCCAGGACGACGAGGCTTGCAGGATCCTCTAGAG60                 TCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGA99                                      GlyAspGlyGlyGly                                                                15                                                                             (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 99 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 61..99                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       GTGTTGCTGCGTTCCCGACCTGCAGCCCAAGCTCTAGAGTCGACCTGCAGCCCAAGCTCA60                 GATCTGCTCATGCTCGCGGCCGCCATGCCCCCGGAAGCG99                                      AspLeuLeuMetLeuAlaAlaAlaMetProProGluAla                                        1510                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 60 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       AGGCAGATCTGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATC60                 (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1386 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bovine herpesvirus-1 (IBR virus)                                 (B) STRAIN: Cooper                                                             (C) INDIVIDUAL ISOLATE: S-IBR- 000                                             (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: 439-18.1 (PSY 1643)                                                 (viii) POSITION IN GENOME:                                                     (B) MAP POSITION:  86.8 to 87.8                                                (C) UNITS: %G                                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 7..1329                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       GCGATCATGCCTGCCGCCCGGACCGGCACCTTGGCCGCCGTCGCCCTA48                             MetProAlaAlaArgThrGlyThrLeuAlaAlaValAlaLeu                                     1510                                                                           ATCCTGCTCTGCGGGGCCGCCGTTTTGCGGCCCCGCGCCCGACGACCT96                             IleLeuLeuCysGlyAlaAlaValLeuArgProArgAlaArgArgPro                               15202530                                                                       CTGTTTCGCCGACGTGCGCCGCACTGGCATGGCGCCCTCCCGCCCGCT144                            LeuPheArgArgArgAlaProHisTrpHisGlyAlaLeuProProAla                               354045                                                                         GGGGCCCGTCCTGAACCTAGCGGCCTCGGATTTGACCTCGCGGGTTTC192                            GlyAlaArgProGluProSerGlyLeuGlyPheAspLeuAlaGlyPhe                               505560                                                                         GGTGCGCGCGGTGGAGCTTCGCGCGCTGCGCCCTGGCCCTCTTGGACA240                            GlyAlaArgGlyGlyAlaSerArgAlaAlaProTrpProSerTrpThr                               657075                                                                         TGGCGGAGACGGTGGTGCCCGGCGGACCGCGAGCCSCACGTCGTCGAC288                            TrpArgArgArgTrpCysProAlaAspArgGluProHisValValAsp                               808590                                                                         GTCGGCTGGGCTTACCAAGACGGGGACTGCATGGTGCCTCTGGCATAT336                            ValGlyTrpAlaTyrGlnAspGlyAspCysMetValProLeuAlaTyr                               95100105110                                                                    CGCCAGTACTTTAACTGCACGGGGGGCGCGCTGCCCGGCCAAAACGTC384                            ArgGlnTyrPheAsnCysThrGlyGlyAlaLeuProGlyGlnAsnVal                               115120125                                                                      TGCGCCGGGCTCTCTGAGACCCGCATCCGCGGTGGCTTTGGAACCTCC432                            CysAlaGlyLeuSerGluThrArgIleArgGlyGlyPheGlyThrSer                               130135140                                                                      GACTACGCGCTCTACGGGACGTCGCTAGTACTGCGCCCCGGCCTGTAC480                            AspTyrAlaLeuTyrGlyThrSerLeuValLeuArgProGlyLeuTyr                               145150155                                                                      GACCGCGGGACCTACATCTACTTCCTTGGATACGGCCCAGACGACATC528                            AspArgGlyThrTyrIleTyrPheLeuGlyTyrGlyProAspAspIle                               160165170                                                                      TACGTGGGCAGCGTCACGCTCATGGTGGGCGCCGACATCCACAAATAC576                            TyrValGlySerValThrLeuMetValGlyAlaAspIleHisLysTyr                               175180185190                                                                   CCCTGCGGGCTGGACCGAGGGCTCGGTGTGGCCCTGCACCACAAGAGC624                            ProCysGlyLeuAspArgGlyLeuGlyValAlaLeuHisHisLysSer                               195200205                                                                      GGACCGGCCCGACCTCTGACAGAGGACGACGCCACCGGCGACTGGGCC672                            GlyProAlaArgProLeuThrGluAspAspAlaThrGlyAspTrpAla                               210215220                                                                      TGCGGCTGCTTCCCCGCCCTTGTTGAGGTTGACGCGGTGTGGGGCAAC720                            CysGlyCysPheProAlaLeuValGluValAspAlaValTrpGlyAsn                               225230235                                                                      GTAAGCGCCGCAGAGCTGGGCCTGGCCGACCCGATCGACTACGCCGAC768                            ValSerAlaAlaGluLeuGlyLeuAlaAspProIleAspTyrAlaAsp                               240245250                                                                      GAAGGGGGTGAGGTCGAAGTGCTCGAGGACGAAGCCGGGAGCGCCAGC816                            GluGlyGlyGluValGluValLeuGluAspGluAlaGlySerAlaSer                               255260265270                                                                   GGAAACCTGCCGCAGGACGACCCCGACCCCGACCTCGCAGATTGCCGG864                            GlyAsnLeuProGlnAspAspProAspProAspLeuAlaAspCysArg                               275280285                                                                      ACCGTCGGGCTCTTTAGCGAAAGCGACATGTTCCGGACCGCCAGCGGG912                            ThrValGlyLeuPheSerGluSerAspMetPheArgThrAlaSerGly                               290295300                                                                      CCCGAATCGCTGCTGATCGGCGCCGTTGCCAAGGACGTCCTGACGGTG960                            ProGluSerLeuLeuIleGlyAlaValAlaLysAspValLeuThrVal                               305310315                                                                      CCCCTCAATCTGCCGCCCGGCCGCTCTTACGAGGCCCTGCGAAACGCA1008                           ProLeuAsnLeuProProGlyArgSerTyrGluAlaLeuArgAsnAla                               320325330                                                                      TCGCTGGAGTGCAACTCCCGCCCGCGCGAGACCGGCGACGCAGCGGTG1056                           SerLeuGluCysAsnSerArgProArgGluThrGlyAspAlaAlaVal                               335340345350                                                                   GTGGTGATGTCTCTCCAGGAGCCCGCTCGCCTCGAGCGCCGCCCCGAT1104                           ValValMetSerLeuGlnGluProAlaArgLeuGluArgArgProAsp                               355360365                                                                      GCCCGCGCCACCGATCCGGAGTTTGGGCTCTTTGGCCTGCCCGATGAC1152                           AlaArgAlaThrAspProGluPheGlyLeuPheGlyLeuProAspAsp                               370375380                                                                      CCCGCCGTGCGCGCGGCATTCTCATCGGCCTCGCGATCGCTCTGCTGG1200                           ProAlaValArgAlaAlaPheSerSerAlaSerArgSerLeuCysTrp                               385390395                                                                      TGCTGCTGTTTCGCTGGTGATCGTGCTCGTCTGCGCCTGCCGGCTCGC1248                           CysCysCysPheAlaGlyAspArgAlaArgLeuArgLeuProAlaArg                               400405410                                                                      CCGCCCAGCCAAGGCTGCGCGACGCCCCGCGCCGCCACGTTCGCCAAG1296                           ProProSerGlnGlyCysAlaThrProArgAlaAlaThrPheAlaLys                               415420425430                                                                   AGCAACCCCGCGTACGAGCCGATGCTCAGCGTCTGATCGCCGGCACCCCACGC1349                      SerAsnProAlaTyrGluProMetLeuSerVal                                              435440                                                                         CGCCCCGACCCCGCTGTCCCGCGTTTACAATAAACAG1386                                      (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bovine Herpesvirus-1 (IBR Virus)                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       ValGlyTrpAlaTyrGlnAspGlyAspCysMetValProLeuAlaTyr                               151015                                                                         ArgGlnTyrPheAsnCysThrGlyGlyAlaLeuProGlyAsnValLeu                               202530                                                                         CysAla                                                                         (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pseudorabies Virus                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       ValAlaTrpPhePheAspGlyGlyHisCysLysValProLeuValHis                               151015                                                                         ArgGluTyrTyrGlyCysProGlyAspAlaMetProSerValGluThr                               202530                                                                         CysThr                                                                         (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Herpes Simplex Virus Type 2                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       ValThrTyrTyrArgLeuThrArgAlaCysArgGlnProIleLeuLeu                               151015                                                                         ArgGlnTyrGlyGlyCysArgGlyGlyGluProProSerProLysThr                               202530                                                                         CysGly                                                                         (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 102 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       CACATACGATTTAGGTGACACTATAGAATACAAGCTTGGGCTGCAGGTCGACTCTAGAGT60                 CGACCTGCAGTGAATAATAAAATGTGTGTTTGTCCGAAATAC102                                  (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 102 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       GCGTTTGAGATTTCTGTCCCGACTAAATTCATGTCGCGCGATAGTGGTGTTTATCGCCGA60                 TAGAGATGGCGATATTGGAAAAATCGATATTTGAAAATATGG102                                  (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 102 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       CATATTGAAAATGTCGCCGATGTGAGTTTCTGTGTAACTGATCGCGTGTTTGGAGGCAAC60                 CGGGGCCTGCTCCCGACGGCCAGCGACGACGTGGTGCTCAAG102                                  (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 102 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..102                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       ATGTCTCTCCAGGAGCCCGCTCGCCTCGAGGGCCTGCCCTCGCAGCTG48                             MetSerLeuGlnGluProAlaArgLeuGluGlyLeuProSerGlnLeu                               151015                                                                         CCCGTCTTCGAGGACACGCAGCGCTACGACGCCTCCCCCGCGTCCGTG96                             ProValPheGluAspThrGlnArgTyrAspAlaSerProAlaSerVal                               202530                                                                         AGCTGG102                                                                      SerTrp                                                                         (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 102 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..42                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 43..63                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 64..102                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       CCCGTGAGCAGCATGATCGTCGTCATCGCCGGCATCGGGATCCTGGCC48                             ProValSerSerMetIleValValIleAlaGlyIleGlyIleLeuAla                               15101                                                                          ATCGTGCTGGTCATCCATATGGCGATCATCAGGGCCCGGGCCCGGAAC96                             IleValLeuValIleHisMetAlaIleIleArgAlaArgAlaArgAsn                               51510                                                                          GACGGC102                                                                      AspGly                                                                         (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 75 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       GGGCCAGTACCGGCGCCTGGTGTCCGTCGACTCTAGAGTCGACCTGCAGCCCAAGCTTTG60                 GCGTAATCATGGTCA75                                                              (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       ACATACGATTTAGGTGACACTATAGAATACAAGCTTAACGAATGAACCGTCGTAAAG57                    (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 96 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..12                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       GTCGAAGTGCTCGAAATTCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTG52                         ValGluValLeu                                                                   1                                                                              CAGGTCGACTCTAGAGGATCTCGACGGACACCAGGCGCCGGTAC96                                 (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       ValGluValLeu                                                                   1                                                                              (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       GGGCGGGGCCGGGTCAGCCGGATCTAGAGTCCCAGGACCCAACGCTGCCCGAGTTTG57                    (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       TCCCAGTCACGACGTTGTAAAACGACGGGATCCATGGTCCCGGTGTCTTCTATGGAG57                    (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 49..57                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       ATTCACTGCAGGTCGACTCTAGAGGATCCCCGGGCGAGCTCGAATTTCGAGCGCCGC57                    GluArgArg                                                                      1                                                                              (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..27                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       GCGCGCGCGTACAACGCCACGGTCATAGGGCGAGCTCGAATTCGTAA47                              AlaArgAlaTyrAsnAlaThrValIle                                                    15                                                                             TCATGGTCAT57                                                                   (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       ATACACATACGATTTAGGTGACACTATAGAATACAAGCTCGCGTGTTTGGAGGCAAC57                    (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 102 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       TCGGGGTAGCCCCAATTCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTGCAGGTCGAC60                 TCTAGAGGATCTCGACGGACACCAGGCGCCGGTACTGGCCCT102                                  (2) INFORMATION FOR SEQ ID NO:35:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 84 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 67..84                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                       CGGGGTAGCCCCAATTCGAGCTCGCCCGGGGATCCTCTAGAGGATCCCCGGGCGAGCTCG60                 AATTTCGAGCGCCGCCCCGATGCC84                                                     GluArgArgProAspAla                                                             15                                                                             (2) INFORMATION FOR SEQ ID NO:36:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2040 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bovine herpesvirus-1 (IBR virus)                                 (B) STRAIN: Cooper                                                             (C) INDIVIDUAL ISOLATE: S-IBR- 000                                             (vii) IMMEDIATE SOURCE:                                                        (B) CLONE:PSY 1644, PSY 1645                                                   (viii) POSITION IN GENOME:                                                     (B) MAP POSITION:  86.8 to 87.8                                                (C) UNITS: %G                                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 85..1935                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       GCGGGCAAGGCGGAGGAAGACCGGGGGCAGGAGCTGCGTGGAGGGCGGAGCCGTTGAGCG60                 GCCCGACCGCCGCCGGGTTGTTAAATGGGTCTCGCGCGGCTCGTGGTTCCA111                         MetGlyLeuAlaArgLeuValValPro                                                    15                                                                             CACCGCGCCGGAGAACCAGCGCGCAGCTTCGCTGCGTGTGTCCCGCGA159                            HisArgAlaGlyGluProAlaArgSerPheAlaAlaCysValProArg                               10152025                                                                       GCTGCGTTCCGGGGAACGGCGCGCGCGAGAGGGTTCGAAAAGGGCATT207                            AlaAlaPheArgGlyThrAlaArgAlaArgGlyPheGluLysGlyIle                               303540                                                                         TGGCAATGCAACCCACCGCGCCGCCCCGGCSSGGTTGCGCCGCTGCTG255                            TrpGlnCysAsnProProArgArgProGlyXxxValAlaProLeuLeu                               455055                                                                         CTGCCGCAGTTATTGCTTTTCGGGCTGATGGCCGAGGCCAAGCCCGCG303                            LeuProGlnLeuLeuLeuPheGlyLeuMetAlaGluAlaLysProAla                               606570                                                                         ACCGAAACCCCGGGCTCGGCTTCGGTCGACACGGTCTTCACGGCGCGC351                            ThrGluThrProGlySerAlaSerValAspThrValPheThrAlaArg                               758085                                                                         GCTGGCGCGCCCGTCTTTCTCCCAGGGCCCGCGGCGCGCCCGGACGTG399                            AlaGlyAlaProValPheLeuProGlyProAlaAlaArgProAspVal                               9095100105                                                                     CGCGCCGTTCGCGGCTGGAGCGTCCTCGCGGCCGCCTGCTCGCCGCCC447                            ArgAlaValArgGlyTrpSerValLeuAlaAlaAlaCysSerProPro                               110115120                                                                      GTGCCGGAGCCCGTCTGCCTCGACGACCGCGAGTGCTTCACCGACGTG495                            ValProGluProValCysLeuAspAspArgGluCysPheThrAspVal                               125130135                                                                      GCCCTGGACGCGGCCTGCCTGCGAACCGCCCGCGTGGCCCCGCTGGCC543                            AlaLeuAspAlaAlaCysLeuArgThrAlaArgValAlaProLeuAla                               140145150                                                                      ATCGCGGAGCTCGCCGAGCGGCCCGACTCAACGGGCGACAAAGAGTTT591                            IleAlaGluLeuAlaGluArgProAspSerThrGlyAspLysGluPhe                               155160165                                                                      GTTCTCGCCGACCCGCACGTCTCGGCGCAGCTGGGTCGCAACGCGACC639                            ValLeuAlaAspProHisValSerAlaGlnLeuGlyArgAsnAlaThr                               170175180185                                                                   GGGGTGCTGATCGCGGCCGCAGCCGAGGAGGACGGCGGCGTGTACTTC687                            GlyValLeuIleAlaAlaAlaAlaGluGluAspGlyGlyValTyrPhe                               190195200                                                                      CTGTACGACCGGCTCATCGGCGACGCCGGCGACGAGGAGACGCAGTTG735                            LeuTyrAspArgLeuIleGlyAspAlaGlyAspGluGluThrGlnLeu                               205210215                                                                      GCGCTGACGCTGCAGGTCGCGACGGCCGGCGCGCAGGGCGCCGCGCGG783                            AlaLeuThrLeuGlnValAlaThrAlaGlyAlaGlnGlyAlaAlaArg                               220225230                                                                      GACGAGGAGAGGGAACCAGCGACCGGGCCCACCCCCGGCCCGCCGCCC831                            AspGluGluArgGluProAlaThrGlyProThrProGlyProProPro                               235240245                                                                      CACCGCACGACGACACGCGCGCCCCCGCGGCGGCACGGCGCGCGCTTC879                            HisArgThrThrThrArgAlaProProArgArgHisGlyAlaArgPhe                               250255260265                                                                   CGCGTGCTGCCGTACCACTCCCACGTATACACCCCGGGCGATTCCTTT927                            ArgValLeuProTyrHisSerHisValTyrThrProGlyAspSerPhe                               270275280                                                                      CTGCTATCGGTGCGTCTGCAGTCTGAGTTTTTCGACGAGGCTCCCTTC975                            LeuLeuSerValArgLeuGlnSerGluPhePheAspGluAlaProPhe                               285290295                                                                      TCGGCCAGCATCGACTGGTACTTCCTGCGGACGGCCGGCGACTGCGCG1023                           SerAlaSerIleAspTrpTyrPheLeuArgThrAlaGlyAspCysAla                               300305310                                                                      CTCATCCGCATATACGAGACGTGCATCTTCCACCCCGAGGCACCGGCC1071                           LeuIleArgIleTyrGluThrCysIlePheHisProGluAlaProAla                               315320325                                                                      TGCCTGCACCCCGCCGACGCGCAGTGCAGCTTCGCGTCGCCGTACCGC1119                           CysLeuHisProAlaAspAlaGlnCysSerPheAlaSerProTyrArg                               330335340345                                                                   TCCGAGACCGTGTACAGCCGGCTGTACGAGCAGTGCCGCCCGGACCCT1167                           SerGluThrValTyrSerArgLeuTyrGluGlnCysArgProAspPro                               350355360                                                                      GCCGGTCGCTGGCCGCACGAGTGCGAGGGCGCCGCGTACGCGGCGCCC1215                           AlaGlyArgTrpProHisGluCysGluGlyAlaAlaTyrAlaAlaPro                               365370375                                                                      GTTGCGCACCTGCGTCCCGCCAATAACAGCGTAGACCTGGTCTTTGAC1263                           ValAlaHisLeuArgProAlaAsnAsnSerValAspLeuValPheAsp                               380385390                                                                      GACGCGCCGGCTGCGGCCTCCGGGCTTTACGTCTTTGTGCTGCAGTAC1311                           AspAlaProAlaAlaAlaSerGlyLeuTyrValPheValLeuGlnTyr                               395400405                                                                      AACGGCCACGTGGAAGCTTGGGACTACTGCCTAGTCGTTACTTCGGAC1359                           AsnGlyHisValGluAlaTrpAspTyrCysLeuValValThrSerAsp                               410415420425                                                                   CGTTTGGTGCGCGCGGTCACCGACCACACGCGCCCCGAGGCCGCAGCC1407                           ArgLeuValArgAlaValThrAspHisThrArgProGluAlaAlaAla                               430435440                                                                      GCCGACGCTCCCGAGCCAGGCCCACCGCTCACCAGCGAGCCGGCGGGG1455                           AlaAspAlaProGluProGlyProProLeuThrSerGluProAlaGly                               445450455                                                                      GSGCCCACCGGGCCCGCGCCCTGGCTTGTGGTGCTGGTGGGCGCGCTT1503                           XxxProThrGlyProAlaProTrpLeuValValLeuValGlyAlaLeu                               460465470                                                                      GGACTCGCGGGACTGGTGGGCATCGCAGCCCTCGCCGTTCGGGTGTGC1551                           GlyLeuAlaGlyLeuValGlyIleAlaAlaLeuAlaValArgValCys                               475480485                                                                      GCGCGCCGCGCAAGCCAGAAGCGCACCTACGACATCCTCAACCCCTTC1599                           AlaArgArgAlaSerGlnLysArgThrTyrAspIleLeuAsnProPhe                               490495500505                                                                   GGGCCCGTATACACCAGCTTGCCGACCAACGAGCCGCTCGACGTGGTG1647                           GlyProValTyrThrSerLeuProThrAsnGluProLeuAspValVal                               510515520                                                                      GTGCCAGTTAGCGACGACGAATTTTCCCTCGACGAAGACTCTTTTGCG1695                           ValProValSerAspAspGluPheSerLeuAspGluAspSerPheAla                               525530535                                                                      GATGACGACAGCGACGATGACGGGCCCGCTAGCAACCCCCCTGCGGAT1743                           AspAspAspSerAspAspAspGlyProAlaSerAsnProProAlaAsp                               540545550                                                                      GCCTACGACCTCGCCGGCGCCCCAGAGCCAACTAGCGGGTTTGCGCGA1791                           AlaTyrAspLeuAlaGlyAlaProGluProThrSerGlyPheAlaArg                               555560565                                                                      GCCCCCGCCAACGGCACGCGCTCGAGTCGCTCTGGGTTCAAAGTTTGG1839                           AlaProAlaAsnGlyThrArgSerSerArgSerGlyPheLysValTrp                               570575580585                                                                   TTTAGGGACCCGCTTGAAGACGATGCCGCGCCAGCGCGGACCCCGGCC1887                           PheArgAspProLeuGluAspAspAlaAlaProAlaArgThrProAla                               590595600                                                                      GCACCAGATTACACCGTGGTAGCAGCGCGACTCAAGTCCATCCTCCGC1935                           AlaProAspTyrThrValValAlaAlaArgLeuLysSerIleLeuArg                               605610615                                                                      TAGGCGCCCCCCCCCGCGCGCTGTGCCGTCTGACGGAAAGCACCCGCGTGTAGGGCTGCA1995               TATAAATGGAGCGCTCACACAAAGCCTCGTGCGGCTGCTTCGAAG2040                              (2) INFORMATION FOR SEQ ID NO:37:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 50 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Herpes Simplex Virus Type 1                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                       TrpLeuArgPheAspValProThrSerCysAlaGluMetArgIleTyr                               151015                                                                         GluSerCysLeuTyrHisProGlnLeuProGluCysLeuSerProAla                               202530                                                                         AspAlaProCysAlaAlaSerThrTrpThrSerArgLeuAlaValArg                               354045                                                                         SerTyr                                                                         50                                                                             (2) INFORMATION FOR SEQ ID NO:38:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 52 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pseudorabies Virus                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                       TrpTyrTyrAlaArgAlaProProArgCysLeuLeuTyrTyrValTyr                               151015                                                                         GluProCysIleTyrHisProArgAlaProGluCysLeuArgProVal                               202530                                                                         AspProAlaCysSerPheThrSerProAlaArgAlaAlaLeuValAla                               354045                                                                         ArgArgAlaTyr                                                                   50                                                                             (2) INFORMATION FOR SEQ ID NO:39:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 52 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Varicella-Zoster Virus                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       TrpLeuTyrValProIleAspProThrCysGlnProMetArgLeuTyr                               151015                                                                         SerThrCysLeuTyrHisProAsnAlaProGlnCysLeuSerHisMet                               202530                                                                         AsnSerGlyCysThrPheThrSerProHisLeuAlaGlnArgValAla                               354045                                                                         SerThrValTyr                                                                   50                                                                             (2) INFORMATION FOR SEQ ID NO:40:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 52 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bovine Herpesvirus-1 (IBR Virus)                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                       TrpTyrPheLeuArgThrAlaGlyAspCysAlaLeuIleArgIleTyr                               151015                                                                         GluThrCysIlePheHisProGluAlaProAlaCysLeuHisProAla                               202530                                                                         AspAlaGlnCysSerPheAlaSerProTyrArgSerGluThrValTyr                               354045                                                                         SerArgLeuTyr                                                                   50                                                                             (2) INFORMATION FOR SEQ ID NO:41:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 84 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 34..84                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                       TTGGGCTGCAGGTCGACTCTAGAGGATCCCCTATGGTACAAGATCGAGAGCGGG54                       TrpTyrLysIleGluSerGly                                                          15                                                                             TGCGCCCGGCCGCTGTACTACATGGAGTAC84                                               CysAlaArgProLeuTyrTyrMetGluTyr                                                 1015                                                                           (2) INFORMATION FOR SEQ ID NO:42:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 84 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..84                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                       TCCGGGCTTTACGTCTTTGTGCTGCAGTACAACGGCCACGTGGAAGCT48                             SerGlyLeuTyrValPheValLeuGlnTyrAsnGlyHisValGluAla                               151015                                                                         TGGGACTACAGCCTAGTCGTTACTTCGGACCGTTTG84                                         TrpAspTyrSerLeuValValThrSerAspArgLeu                                           2025                                                                           (2) INFORMATION FOR SEQ ID NO:43:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 84 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                       CCTTCACCGCCGCCGGAAGGCTCCATCGTGTCCATCCCCATCCTCGAGCTCGAATTGGGG60                 ATCCTCTAGAGTCGACCTGCAGCC84                                                     (2) INFORMATION FOR SEQ ID NO:44:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 60 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 28..33                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                       CTATAGAATACACGGAATTCGAGCTCGCCCGGGTGAGCGGCCTAGGCCCTCCC53                        ProGly                                                                         1                                                                              CCGACCG60                                                                      (2) INFORMATION FOR SEQ ID NO:45:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 90 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..33                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                       ATGGCCGAGGCCAAGCCCGCGACCGAAACCCCGGGGATCCTCTAGAGTCGACG53                        MetAlaGluAlaLysProAlaThrGluThrPro                                              1510                                                                           TCTGGGGCGCGGGGGTGGTGCTCTTCGAGACGCTGCC90                                        (2) INFORMATION FOR SEQ ID NO:46:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 90 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 28..48                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 49..90                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                       ACCTTTGCGCATCTCCACAGCTCAACAATGAAGTGGGCAACGTGGATCGAT51                          MetLysTrpAlaThrTrpIleAsp                                                       151                                                                            CCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC90                                      ProValValLeuGlnArgArgAspTrpGluAsnProGly                                        510                                                                            (2) INFORMATION FOR SEQ ID NO:47:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 216 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..84                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 134..190                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                       TGGAGCCCGTCAGTATCGGCGGAAATCCAGCTGAGCGCCGGTCGCTAC48                             TrpSerProSerValSerAlaGluIleGlnLeuSerAlaGlyArgTyr                               151015                                                                         CATTACCAGTTGGTCTGGTGTCAAAAAGATCTAGAATAAGCTAGAG94                               HisTyrGlnLeuValTrpCysGlnLysAspLeuGlu                                           2025                                                                           GATCGATCCCCTATGGCGATCATCAGGGCCCGATCCCCTATGGCGATCATCAGG148                      MetAlaIleIleArg                                                                15                                                                             GCCCGGGCCCGGAACGACGGCTACCGCCACGTGGCCTCCGCC190                                  AlaArgAlaArgAsnAspGlyTyrArgHisValAlaSerAla                                     1015                                                                           TGACCCGGCCCCGCCCGACTCCCCCG216                                                  (2) INFORMATION FOR SEQ ID NO:48:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 90 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 49..90                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                       GGCGCCTGGTGTCCGTCGACTCTAGAGTCGACCTGCAGCCCAAGCTCTAGCAACCCC57                    SerAsnPro                                                                      1                                                                              CCTGCGGATGCCTACGACCTCGCCGGCGCCCCA90                                            ProAlaAspAlaTyrAspLeuAlaGlyAlaPro                                              510                                                                            (2) INFORMATION FOR SEQ ID NO:49:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1880 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Parainfluenza-3 virus                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 73..1788                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                       AGGAACAAAGTTGTTCAACACAGCAGCAGCGAACAGACCCAAAGGCAGCGCAGAGGCGAC60                 ACCGAACCCAAAATGGAATATTGGAAACACACAAACAGCACAAAAAAC108                            MetGluTyrTrpLysHisThrAsnSerThrLysAsn                                           1510                                                                           ACCAACAATGAAACCGAAACAACCAGAGGCAAACACAGTAGCAAGGTT156                            ThrAsnAsnGluThrGluThrThrArgGlyLysHisSerSerLysVal                               152025                                                                         ACAAATATCATAATGTACACCTTCTGGACAATAACATCAACAATATTA204                            ThrAsnIleIleMetTyrThrPheTrpThrIleThrSerThrIleLeu                               303540                                                                         TTAGTCATTTTTATAATGATATTGACAAACTTAATTCAAGAGAACAAT252                            LeuValIlePheIleMetIleLeuThrAsnLeuIleGlnGluAsnAsn                               45505560                                                                       CATAATAAATTAATGTTGCAGGAAATAAGAAAAGAATTCGCGGCAATA300                            HisAsnLysLeuMetLeuGlnGluIleArgLysGluPheAlaAlaIle                               657075                                                                         GACACCAAGATTCAGAGGACCTCGGATGACATTGGAACCTCAATACAG348                            AspThrLysIleGlnArgThrSerAspAspIleGlyThrSerIleGln                               808590                                                                         TCAGGAATAAATACAAGACTTCTCACAATTCAGAGTCATGTTCAAAAC396                            SerGlyIleAsnThrArgLeuLeuThrIleGlnSerHisValGlnAsn                               95100105                                                                       TATATCCCACTATCACTAACACAACAAATGTCAGATCTCAGAAAATTT444                            TyrIleProLeuSerLeuThrGlnGlnMetSerAspLeuArgLysPhe                               110115120                                                                      ATCAATGATCTAACAAATAAAAGAGAACATCAAGAAGTGCCAATACAG492                            IleAsnAspLeuThrAsnLysArgGluHisGlnGluValProIleGln                               125130135140                                                                   AGAATGACTCATGATAGAGGTATAGAACCCCTAAATCCAGACAAGTTC540                            ArgMetThrHisAspArgGlyIleGluProLeuAsnProAspLysPhe                               145150155                                                                      TGGAGGTGTACATCTGGTAACCCATCTCTAACAAGTAGTCCTAAGATA588                            TrpArgCysThrSerGlyAsnProSerLeuThrSerSerProLysIle                               160165170                                                                      AGGTTAATACCAGGGCCAGGTTTATTAGCAACATCTACTACAGTAAAT636                            ArgLeuIleProGlyProGlyLeuLeuAlaThrSerThrThrValAsn                               175180185                                                                      GGCTGTATTAGAATCCCATCGTTAGCAATCAATCATTTAATCTACGCT684                            GlyCysIleArgIleProSerLeuAlaIleAsnHisLeuIleTyrAla                               190195200                                                                      TACACCTCTAATCTTATCACCCAGGGCTGTCAAAATATAGGGAAATCT732                            TyrThrSerAsnLeuIleThrGlnGlyCysGlnAsnIleGlyLysSer                               205210215220                                                                   TACCAAGTACTACAAATAGGGATAATTACTATAAATTCGGACCTAGTA780                            TyrGlnValLeuGlnIleGlyIleIleThrIleAsnSerAspLeuVal                               225230235                                                                      CCTGATTTAAATCCCAGAGTCACACATACATTTAATATTGATGATAAT828                            ProAspLeuAsnProArgValThrHisThrPheAsnIleAspAspAsn                               240245250                                                                      AGGAAATCTTGCTCTCTGGCACTATTGAATACAGATGTTTATCAGTTA876                            ArgLysSerCysSerLeuAlaLeuLeuAsnThrAspValTyrGlnLeu                               255260265                                                                      TGCTCAACACCAAAAGTTGATGAGAGATCCGATTATGCATCAACAGGT924                            CysSerThrProLysValAspGluArgSerAspTyrAlaSerThrGly                               270275280                                                                      ATTGAGGATATTGTACTTGACATTGTCACTAATAATGGATTAATTATA972                            IleGluAspIleValLeuAspIleValThrAsnAsnGlyLeuIleIle                               285290295300                                                                   ACAACAAGGTTTACAAATAATAATATAACTTTTGATAAACCGTATGCA1020                           ThrThrArgPheThrAsnAsnAsnIleThrPheAspLysProTyrAla                               305310315                                                                      GCATTGTATCCATCAGTAGGACCAGGAATCTATTATAAGGGTAAAGTT1068                           AlaLeuTyrProSerValGlyProGlyIleTyrTyrLysGlyLysVal                               320325330                                                                      ATCTTTCTCGGATATGGAGGTCTAGAGCATGAAGAAAACGGAGACGTA1116                           IlePheLeuGlyTyrGlyGlyLeuGluHisGluGluAsnGlyAspVal                               335340345                                                                      ATATGTAATACAACTGGTTGTCCTGGCAAAACACAGAGAGACTGTAAT1164                           IleCysAsnThrThrGlyCysProGlyLysThrGlnArgAspCysAsn                               350355360                                                                      CAGGCTTCTTATAGCCCATGGTTCTCAAATAGGAGAATGGTAAACTCT1212                           GlnAlaSerTyrSerProTrpPheSerAsnArgArgMetValAsnSer                               365370375380                                                                   ATTATTGTTGTTGATAAAGGCATAGATGCAACTTTTAGCTTGAGGGTG1260                           IleIleValValAspLysGlyIleAspAlaThrPheSerLeuArgVal                               385390395                                                                      TGGACTATTCCAATGAGCCAAAATTATTGGGGATCAGAAGGAAGATTA1308                           TrpThrIleProMetSerGlnAsnTyrTrpGlySerGluGlyArgLeu                               400405410                                                                      CTTTTATTAGGTGACAGAATATACATATATACTAGATCCACAAGTTGG1356                           LeuLeuLeuGlyAspArgIleTyrIleTyrThrArgSerThrSerTrp                               415420425                                                                      CACAGTAAATTACAGTTAGGGGTAATTGATATTTCTGATTATAATAAT1404                           HisSerLysLeuGlnLeuGlyValIleAspIleSerAspTyrAsnAsn                               430435440                                                                      ATAAGAATAAATTGGACTTGGCATAATGTACCATCACGGCCAGGAAAT1452                           IleArgIleAsnTrpThrTrpHisAsnValProSerArgProGlyAsn                               445450455460                                                                   GATGAATGTCCATGGGGTCATTCATGCCCAGACGGATGTATAACAGGA1500                           AspGluCysProTrpGlyHisSerCysProAspGlyCysIleThrGly                               465470475                                                                      GTTTACACTGATGCATATCCGCTAAACCCATCGGGGAGTGTTGTATCA1548                           ValTyrThrAspAlaTyrProLeuAsnProSerGlySerValValSer                               480485490                                                                      TCAGTAATTCTTGACTCACAAAAGTCTAGAGAAAACCCAATCATTACC1596                           SerValIleLeuAspSerGlnLysSerArgGluAsnProIleIleThr                               495500505                                                                      TACTCAACAGCTACAAATAGAATAAATGAATTAGCTATATATAACAGA1644                           TyrSerThrAlaThrAsnArgIleAsnGluLeuAlaIleTyrAsnArg                               510515520                                                                      ACACTTCCAGCTGCATATACAACAACAAATTGTATCACACATTATGAT1692                           ThrLeuProAlaAlaTyrThrThrThrAsnCysIleThrHisTyrAsp                               525530535540                                                                   AAAGGGTATTGTTTTCATATAGTAGAAATAAATCACAGAAGTTTGAAT1740                           LysGlyTyrCysPheHisIleValGluIleAsnHisArgSerLeuAsn                               545550555                                                                      ACGTTTCAACCTATGTTATTCAAAACAGAAGTTCCAAAAAACTGCAGC1788                           ThrPheGlnProMetLeuPheLysThrGluValProLysAsnCysSer                               560565570                                                                      TAAATGATCATCGCATATCGGATGCCAGATGACATTAAAAGAGACCACCAGACAGACAAC1848               ACAGGAGATGATGCAAGATATAAAGGAATAAT1880                                           (2) INFORMATION FOR SEQ ID NO:50:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                       GGGAATTCTGCAGGTCACATCATACAATTCTAATCTAAG39                                      (2) INFORMATION FOR SEQ ID NO:51:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 43 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                       GGGAATTCTGCAGGCTTTAAAAGAGAGAATTTCCGTTTGGCTA43                                  __________________________________________________________________________ 

What is claimed is:
 1. A method of distinguishing a bovine vaccinated with a vaccine for infectious bovine rhinotracheitis disease in which the vaccine comprises a) an effective immunizing amount of a live recombinant infectious bovine rhinotracheitis virus comprising an infectious bovine rhinotracheitis viral genome from which DNA encoding the US2 gene and the glycoprotein G gene have been deleted and b) a suitable carrier, from an animal infected with a wild-type infectious bovine rhinotracheitis virus, the method comprising detecting the presence or absence of infectious bovine rhinotracheitis glycoprotein G in a body fluid of the bovine.
 2. A method of distinguishing a bovine vaccinated with a vaccine for infectious bovine rhinotracheitis disease in which the vaccine comprises an effective immunizing amount of a live recombinant infectious bovine rhinotracheitis designated S-IBR-037 (ATCC ACCESSION NO. VR 2320) and a suitable carrier, from an animal infected with a wild-type infectious bovine rhinotracheitis virus, the method comprising detecting the presence or absence of infectious bovine rhinotracheitis glycoprotein G in a body fluid of the bovine.
 3. The method of claim 1 or 2, wherein the presence or absence of the glycoprotein G in the body fluid is determined by detecting in the body fluid antibodies specific for glycoprotein G.
 4. A method of distinguishing a bovine vaccinated with a vaccine for infectious bovine rhinotracheitis disease in which the vaccine comprises a) an effective immunizing amount of a live recombinant infectious bovine rhinotracheitis virus comprising an infectious bovine rhinotracheitis viral genome from which DNA encoding the US2 gene and the glycoprotein E gene has been deleted and b) a suitable carrier, from an animal infected with a wild-type infectious bovine rhinotracheitis virus, the method comprising detecting the presence or absence of infectious bovine rhinotracheitis glycoprotein E in a body fluid of the bovine.
 5. The method of claim 4, wherein the presence or absence of the glycoprotein E in the body fluid is determined by detecting in the body fluid antibodies specific for glycoprotein E. 